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Sample records for schwann cell cytoplasm

  1. Autophagy is involved in the reduction of myelinating Schwann cell cytoplasm during myelin maturation of the peripheral nerve.

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    So Young Jang

    Full Text Available Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.

  2. Schwann cell myelination requires Dynein function

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    Langworthy Melissa M

    2012-11-01

    Full Text Available Abstract Background Interaction of Schwann cells with axons triggers signal transduction that drives expression of Pou3f1 and Egr2 transcription factors, which in turn promote myelination. Signal transduction appears to be mediated, at least in part, by cyclic adenosine monophosphate (cAMP because elevation of cAMP levels can stimulate myelination in the absence of axon contact. The mechanisms by which the myelinating signal is conveyed remain unclear. Results By analyzing mutations that disrupt myelination in zebrafish, we learned that Dynein cytoplasmic 1 heavy chain 1 (Dync1h1, which functions as a motor for intracellular molecular trafficking, is required for peripheral myelination. In dync1h1 mutants, Schwann cell progenitors migrated to peripheral nerves but then failed to express Pou3f1 and Egr2 or make myelin membrane. Genetic mosaic experiments revealed that robust Myelin Basic Protein expression required Dync1h1 function within both Schwann cells and axons. Finally, treatment of dync1h1 mutants with a drug to elevate cAMP levels stimulated myelin gene expression. Conclusion Dync1h1 is required for retrograde transport in axons and mutations of Dync1h1 have been implicated in axon disease. Our data now provide evidence that Dync1h1 is also required for efficient myelination of peripheral axons by Schwann cells, perhaps by facilitating signal transduction necessary for myelination.

  3. Biology of Schwann cells.

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    Kidd, Grahame J; Ohno, Nobuhiko; Trapp, Bruce D

    2013-01-01

    The fundamental roles of Schwann cells during peripheral nerve formation and regeneration have been recognized for more than 100 years, but the cellular and molecular mechanisms that integrate Schwann cell and axonal functions continue to be elucidated. Derived from the embryonic neural crest, Schwann cells differentiate into myelinating cells or bundle multiple unmyelinated axons into Remak fibers. Axons dictate which differentiation path Schwann cells follow, and recent studies have established that axonal neuregulin1 signaling via ErbB2/B3 receptors on Schwann cells is essential for Schwann cell myelination. Extracellular matrix production and interactions mediated by specific integrin and dystroglycan complexes are also critical requisites for Schwann cell-axon interactions. Myelination entails expansion and specialization of the Schwann cell plasma membrane over millimeter distances. Many of the myelin-specific proteins have been identified, and transgenic manipulation of myelin genes have provided novel insights into myelin protein function, including maintenance of axonal integrity and survival. Cellular events that facilitate myelination, including microtubule-based protein and mRNA targeting, and actin based locomotion, have also begun to be understood. Arguably, the most remarkable facet of Schwann cell biology, however, is their vigorous response to axonal damage. Degradation of myelin, dedifferentiation, division, production of axonotrophic factors, and remyelination all underpin the substantial regenerative capacity of the Schwann cells and peripheral nerves. Many of these properties are not shared by CNS fibers, which are myelinated by oligodendrocytes. Dissecting the molecular mechanisms responsible for the complex biology of Schwann cells continues to have practical benefits in identifying novel therapeutic targets not only for Schwann cell-specific diseases but other disorders in which axons degenerate. Copyright © 2013 Elsevier B.V. All rights

  4. Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

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    Sobue, G.; Pleasure, D.

    1985-01-01

    Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125 I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more

  5. Electrically induced release of acetylcholine from denervated Schwann cells.

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    Dennis, M J; Miledi, R

    1974-03-01

    1. Focal electrical stimulation of Schwann cells at the end-plates of denervated frog muscles elicited slow depolarizations of up to 30 mV in the muscle fibres. This response is referred to as a Schwann-cell end-plate potential (Schwann-e.p.p.).2. Repeated stimulation sometimes evoked further Schwann-e.p.p.s, but they were never sustained for more than 30 pulses. Successive e.p.p.s varied in amplitude and time course independently of the stimulus.3. The Schwann-e.p.p.s were reversibly blocked by curare, suggesting that they result from a release of acetylcholine (ACh) by the Schwann cells.4. ACh release by electrical stimulation did not seem to occur in quantal form and was not dependent on the presence of calcium ions in the external medium; nor was it blocked by tetrodotoxin.5. Stimulation which caused release of ACh also resulted in extensive morphological disruption of the Schwann cells, as seen with both light and electron microscopy.6. It is concluded that electrical stimulation of denervated Schwann cells causes break-down of the cell membrane and releases ACh, presumably in molecular form.

  6. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

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    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  7. Axon-Schwann cell interaction in the squid nerve fibre.

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    Villegas, J

    1972-09-01

    The electrical properties of Schwann cells and the effects of neuronal impulses on their membrane potential have been studied in the giant nerve fibre of the squid.1. The behaviour of the Schwann cell membrane to current injection into the cell was ohmic. No impulse-like responses were observed with displacements of 35 mV in the membrane potential. The resistance of the Schwann cell membrane was found to be approximately 10(3) Omega cm(2).2. A long-lasting hyperpolarization is observed in the Schwann cells following the conduction of impulse trains by the axon. Whereas the propagation of a single impulse had little effect, prolonged stimulation of the fibre at 250 impulses/sec was followed by a hyperpolarization of the Schwann cell that gradually declined over a period of several minutes.3. The prolonged effects of nerve impulse trains on the Schwann cell were similar to those produced by depolarizing current pulses applied to the axon by the voltage-clamp technique. Thus, a series of depolarizing pulses in the axon was followed by a long-lasting hyperpolarization of the Schwann cells. In contrast, the application of a series of hyperpolarizing 100 mV pulses at a frequency of 1/sec had no apparent effects.4. Changes in the external potassium concentration did not reproduce the long-lasting effects of nerve excitation.5. The hyperpolarizing effects of impulse trains were abolished by the incubation of the nerve fibre in a sea-water solution containing trypsin.6. These findings are discussed in relation to the possible mechanisms that might be responsible for the long-lasting hyperpolarizations of the Schwann cells.

  8. Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

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    Shu-quan Zhang

    2015-01-01

    Full Text Available Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T 9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined withSchwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.

  9. Schwann Cell Glycogen Selectively Supports Myelinated Axon Function

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    Brown, Angus M; Evans, Richard D; Black, Joel; Ransom, Bruce R

    2012-01-01

    Objectives Interruption of energy supply to peripheral axons is a cause of axon loss. We determined if glycogen was present in mammalian peripheral nerve, and if it supported axon conduction during aglycemia. Methods We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. Results Glycogen was present in sciatic nerve, its concentration varying directly with ambient [glucose]. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time-course of glycogen loss. Latency to CAP failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. Interpretation Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. PMID:23034913

  10. Macrophage polarization in nerve injury: do Schwann cells play a role?

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    Jo Anne Stratton

    2016-01-01

    Full Text Available In response to peripheral nerve injury, the inflammatory response is almost entirely comprised of infiltrating macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris and regulating the microenvironment to allow for efficient regeneration. There are several cells within the microenvironment that likely interact with macrophages to support their function - most notably the Schwann cell, the glial cell of the peripheral nervous system. Schwann cells express several ligands that are known to interact with receptors expressed by macrophages, yet the effects of Schwann cells in regulating macrophage phenotype remains largely unexplored. This review discusses macrophages in peripheral nerve injury and how Schwann cells may regulate their behavior.

  11. Cthrc1 is a negative regulator of myelination in Schwann cells.

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    Apra, Caroline; Richard, Laurence; Coulpier, Fanny; Blugeon, Corinne; Gilardi-Hebenstreit, Pascale; Vallat, Jean-michel; Lindner, Volkhard; Charnay, Patrick; Decker, Laurence

    2012-03-01

    The analysis of the molecular mechanisms involved in the initial interaction between neurons and Schwann cells is a key issue in understanding the myelination process. We recently identified Cthrc1 (Collagen triple helix repeat containing 1) as a gene upregulated in Schwann cells upon interaction with the axon. Cthrc1 encodes a secreted protein previously shown to be involved in migration and proliferation in different cell types. We performed a functional analysis of Cthrc1 in Schwann cells by loss-of- and gain-of-function approaches using RNA interference knockdown in cell culture and a transgenic mouse line that overexpresses the gene. This work establishes that Cthrc1 enhances Schwann cell proliferation but prevents myelination. In particular, time-course analysis of myelin formation intransgenic animals reveals that overexpression of Cthrc1 in Schwann cells leads to a delay in myelin formation with cells maintaining a proliferative state. Our data, therefore, demonstrate that Cthrc1 plays a negative regulatory role, fine-tuning the onset of peripheral myelination.

  12. Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

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    Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

    2017-06-06

    Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Biphasic electrical targeting plays a significant role in schwann cell activation.

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    Kim, In Sook; Song, Yun Mi; Cho, Tae Hyung; Pan, Hui; Lee, Tae Hyung; Kim, Sung June; Hwang, Soon Jung

    2011-05-01

    Electrical stimulation (ES) is a promising technique for axonal regeneration of peripheral nerve injuries. However, long-term, continuous ES in the form of biphasic electric current (BEC) to stimulate axonal regeneration has rarely been attempted and the effects of BEC on Schwann cells are unknown. We hypothesized that long-term, continuous ES would trigger the activation of Schwann cells, and we therefore investigated the effect of BEC on the functional differentiation of primary human mesenchymal stromal cells (hMSCs) into Schwann cells, as well as the activity of primary Schwann cells. Differentiation of hMSCs into Schwann cells was determined by coculture with rat pheochromocytoma cells (PC12 cell line). We also investigated the in vivo effects of long-term ES (4 weeks) on axonal outgrowth of a severed sciatic nerve with a 7-mm gap after retraction of the nerve ends in rats by implanting an electronic device to serve as a neural conduit. PC12 cells cocultured with hMSCs electrically stimulated during culture in Schwann cell differentiation medium (Group I) had longer neurites and a greater percentage of PC12 cells were neurite-sprouting than when cocultured with hMSCs cultured in growth medium (control group) or unstimulated hMSCs in the same culture conditions as used for Group I (Group II). Group I cells showed significant upregulation of Schwann cell-related neurotrophic factors such as nerve growth factor and glial-derived neurotrophic factor compared to Group II cells at both the mRNA and protein levels. Primary Schwann cells responded to continuous BEC with increased proliferation and the induction of nerve growth factor and glial-derived neurotrophic factor, similar to Group I cells, and in addition, induction of brain-derived neurotrophic factor was observed. Immunohistochemical investigation of sciatic nerve regenerates revealed that BEC increased axonal outgrowth significantly. These results demonstrate that BEC enhanced the functional activity of

  14. PAR1 activation affects the neurotrophic properties of Schwann cells.

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    Pompili, Elena; Fabrizi, Cinzia; Somma, Francesca; Correani, Virginia; Maras, Bruno; Schininà, Maria Eugenia; Ciraci, Viviana; Artico, Marco; Fornai, Francesco; Fumagalli, Lorenzo

    2017-03-01

    Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Electrical stimulation of schwann cells promotes sustained increases in neurite outgrowth.

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    Koppes, Abigail N; Nordberg, Andrea L; Paolillo, Gina M; Goodsell, Nicole M; Darwish, Haley A; Zhang, Linxia; Thompson, Deanna M

    2014-02-01

    Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal growth after injury (McCaig 1991, Al-Majed 2000). However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal growth. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite outgrowth and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, fibroblasts] will be treated and this biophysical stimuli can influence axonal growth directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0-200 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite outgrowth relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a 20% increase in total neurite outgrowth and was sustained for 72 h poststimulation. An 11-fold increase in nerve growth factor but not brain-derived neurotrophic factor or glial-derived growth factor was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in fibroblast or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in

  16. After Nerve Injury, Lineage Tracing Shows That Myelin and Remak Schwann Cells Elongate Extensively and Branch to Form Repair Schwann Cells, Which Shorten Radically on Remyelination.

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    Gomez-Sanchez, Jose A; Pilch, Kjara S; van der Lans, Milou; Fazal, Shaline V; Benito, Cristina; Wagstaff, Laura J; Mirsky, Rhona; Jessen, Kristjan R

    2017-09-13

    There is consensus that, distal to peripheral nerve injury, myelin and Remak cells reorganize to form cellular columns, Bungner's bands, which are indispensable for regeneration. However, knowledge of the structure of these regeneration tracks has not advanced for decades and the structure of the cells that form them, denervated or repair Schwann cells, remains obscure. Furthermore, the origin of these cells from myelin and Remak cells and their ability to give rise to myelin cells after regeneration has not been demonstrated directly, although these conversions are believed to be central to nerve repair. Using genetic lineage-tracing and scanning-block face electron microscopy, we show that injury of sciatic nerves from mice of either sex triggers extensive and unexpected Schwann cell elongation and branching to form long, parallel processes. Repair cells are 2- to 3-fold longer than myelin and Remak cells and 7- to 10-fold longer than immature Schwann cells. Remarkably, when repair cells transit back to myelinating cells, they shorten ∼7-fold to generate the typically short internodes of regenerated nerves. The present experiments define novel morphological transitions in injured nerves and show that repair Schwann cells have a cell-type-specific structure that differentiates them from other cells in the Schwann cell lineage. They also provide the first direct evidence using genetic lineage tracing for two basic assumptions in Schwann cell biology: that myelin and Remak cells generate the elongated cells that build Bungner bands in injured nerves and that such cells can transform to myelin cells after regeneration. SIGNIFICANCE STATEMENT After injury to peripheral nerves, the myelin and Remak Schwann cells distal to the injury site reorganize and modify their properties to form cells that support the survival of injured neurons, promote axon growth, remove myelin-associated growth inhibitors, and guide regenerating axons to their targets. We show that the

  17. Pluripotent Stem Cells for Schwann Cell Engineering

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

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

  18. The Wound Microenvironment Reprograms Schwann Cells to Invasive Mesenchymal-like Cells to Drive Peripheral Nerve Regeneration.

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    Clements, Melanie P; Byrne, Elizabeth; Camarillo Guerrero, Luis F; Cattin, Anne-Laure; Zakka, Leila; Ashraf, Azhaar; Burden, Jemima J; Khadayate, Sanjay; Lloyd, Alison C; Marguerat, Samuel; Parrinello, Simona

    2017-09-27

    Schwann cell dedifferentiation from a myelinating to a progenitor-like cell underlies the remarkable ability of peripheral nerves to regenerate following injury. However, the molecular identity of the differentiated and dedifferentiated states in vivo has been elusive. Here, we profiled Schwann cells acutely purified from intact nerves and from the wound and distal regions of severed nerves. Our analysis reveals novel facets of the dedifferentiation response, including acquisition of mesenchymal traits and a Myc module. Furthermore, wound and distal dedifferentiated Schwann cells constitute different populations, with wound cells displaying increased mesenchymal character induced by localized TGFβ signaling. TGFβ promotes invasion and crosstalks with Eph signaling via N-cadherin to drive collective migration of the Schwann cells across the wound. Consistently, Tgfbr2 deletion in Schwann cells resulted in misdirected and delayed reinnervation. Thus, the wound microenvironment is a key determinant of Schwann cell identity, and it promotes nerve repair through integration of multiple concerted signals. VIDEO ABSTRACT. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects.

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    Zong, Haibin; Zhao, Hongxing; Zhao, Yilei; Jia, Jingling; Yang, Libin; Ma, Chao; Zhang, Yang; Dong, Yuzhen

    2013-05-15

    Schwann cells and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this study, we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes. Results showed that neurotrophin-3 was successfully transfected into Schwann cells, where it was expressed effectively and steadily. A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects. Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination, promote nerve axonal and myelin regeneration, and delay apoptosis of spinal motor neurons. Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.

  20. Ribosomal trafficking is reduced in Schwann cells following induction of myelination

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    James M. Love

    2015-08-01

    Full Text Available Local synthesis of proteins within the Schwann cell periphery is extremely important for efficient process extension and myelination, when cells undergo dramatic changes in polarity and geometry. Still, it is unclear how ribosomal distributions are developed and maintained within Schwann cell projections to sustain local translation. In this multi-disciplinary study, we expressed a plasmid encoding a fluorescently labeled ribosomal subunit (L4-GFP in cultured primary rat Schwann cells. This enabled the generation of high-resolution, quantitative data on ribosomal distributions and trafficking dynamics within Schwann cells during early stages of myelination, induced by ascorbic acid treatment. Ribosomes were distributed throughout Schwann cell projections, with ~2-3 bright clusters along each projection. Clusters emerged within 1 day of culture and were maintained throughout early stages of myelination. Three days after induction of myelination, net ribosomal movement remained anterograde (directed away from the Schwann cell body, but ribosomal velocity decreased to about half the levels of the untreated group. Statistical and modeling analysis provided additional insight into key factors underlying ribosomal trafficking. Multiple regression analysis indicated that net transport at early time points was dependent on anterograde velocity, but shifted to dependence on anterograde duration at later time points. A simple, data-driven rate kinetics model suggested that the observed decrease in net ribosomal movement was primarily dictated by an increased conversion of anterograde particles to stationary particles, rather than changes in other directional parameters. These results reveal the strength of a combined experimental and theoretical approach in examining protein localization and transport, and provide evidence of an early establishment of ribosomal populations within Schwann cell projections with a reduction in trafficking following

  1. Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves

    NARCIS (Netherlands)

    Gomez-Sanchez, Jose A.; Carty, Lucy; Iruarrizaga-Lejarreta, Marta; Palomo-Irigoyen, Marta; Varela-Rey, Marta; Griffith, Megan; Hantke, Janina; Macias-Camara, Nuria; Azkargorta, Mikel; Aurrekoetxea, Igor; de Juan, Virginia Gutiérrez; Jefferies, Harold B. J.; Aspichueta, Patricia; Elortza, Félix; Aransay, Ana M.; Martínez-Chantar, María L.; Baas, Frank; Mato, José M.; Mirsky, Rhona; Woodhoo, Ashwin; Jessen, Kristján R.

    2015-01-01

    Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell-mediated myelin digestion possible have not been established. We report that

  2. Electrical regulation of Schwann cells using conductive polypyrrole/chitosan polymers.

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    Huang, Jinghui; Hu, Xueyu; Lu, Lei; Ye, Zhengxu; Zhang, Quanyu; Luo, Zhuojing

    2010-04-01

    Electrical stimulation (ES) can dramatically enhance neurite outgrowth through conductive polymers and accelerate peripheral nerve regeneration in animal models of nerve injury. Therefore, conductive tissue engineering graft in combination with ES is a potential treatment for neural injuries. Conductive tissue engineering graft can be obtained by seeding Schwann cells on conductive scaffold. However, when ES is applied through the conductive scaffold, the impact of ES on Schwann cells has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate Schwann cells. The tolerance of Schwann cells to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a MTT assay. mRNA and protein levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in Schwann cells were assayed by RT-PCR and Western blotting, and the amount of NGF and BDNF secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of NGF and BDNF when compared with control cells without ES. These findings highlight for the first time the possibility of enhancing nerve regeneration in conductive scaffolds through ES-increased neurotrophin secretion.

  3. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

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    Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

    2013-01-01

    Highlights: ► ATP-treated sciatic explants shows the decreased expression of p75NGFR. ► Extracellular ATP inhibits the expression of phospho-ERK1/2. ► Lysosomal exocytosis is involved in Schwann cell dedifferentiation. ► Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  4. Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration.

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    Yamamoto, Tsubasa; Osako, Yohei; Ito, Masataka; Murakami, Masashi; Hayashi, Yuki; Horibe, Hiroshi; Iohara, Koichiro; Takeuchi, Norio; Okui, Nobuyuki; Hirata, Hitoshi; Nakayama, Hidenori; Kurita, Kenichi; Nakashima, Misako

    2016-01-01

    Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

  5. GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves.

    Science.gov (United States)

    May, Florian; Matiasek, Kaspar; Vroemen, Maurice; Caspers, Christiane; Mrva, Thomas; Arndt, Christian; Schlenker, Boris; Gais, Peter; Brill, Thomas; Buchner, Alexander; Blesch, Armin; Hartung, Rudolf; Stief, Christian; Gansbacher, Bernd; Weidner, Norbert

    2008-11-01

    Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats. Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves). The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p=0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls. These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.

  6. Neuron-glia signaling and the protection of axon function by Schwann cells.

    Science.gov (United States)

    Quintes, Susanne; Goebbels, Sandra; Saher, Gesine; Schwab, Markus H; Nave, Klaus-Armin

    2010-03-01

    The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron-Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt-Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.

  7. Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

    International Nuclear Information System (INIS)

    Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji

    2007-01-01

    Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS

  8. Proliferation of Schwann cells induced by axolemmal and myelin membranes

    International Nuclear Information System (INIS)

    Dinneen, M.

    1985-01-01

    Purified Schwann Cells were cultured from neonatal rat sciatic nerve using a modification of the method of Brockes. Schwann cells and contaminating fibroblasts were unambiguously identified using fluorescent antibodies of 2'3' cyclic nucleotide 3'-phosphodiesterase and the thy 1.1 antigen respectively. The Schwann cells were quiescent unless challenged with mitogens. They proliferated rapidly in response to the soluble mitogen, cholera toxin, or to membrane fractions from rat CNS or PNS, prepared by the method of DeVries. Mitogenic activity was present in both axolemmal and myelin enriched fractions and promoted a 10-15 fold increase in the rate of 3 H-thymidine uptake. The axolemmal mitogen was sensitive to heat (80 0 C for 10 minutes), trypsin digestion (0.05% x 30 mins) or to treatment with endoglycosidase D, suggesting that it could be a glycoprotein. Fifty percent of the axolemmal mitogenic activity was solubilized in 1% octyl-glucoside. The solubilized material, however, was very unstable and further purification was not possible. The myelin associated mitogenic activity was markedly different. It was resistant to freeze thaw cycles, trypsin digestion of endoglycosidase treatment and the activity was actually enhanced by heating at 100 0 C for two hours. It is proposed that the axolemmal activity is responsible for Schwann cell proliferation during development and that the myelin associated activity promotes Schwann cell proliferation during Wallerian degeneration

  9. STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

    Science.gov (United States)

    Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

    2017-04-19

    After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal

  10. Liposomes to target peripheral neurons and Schwann cells.

    Directory of Open Access Journals (Sweden)

    Sooyeon Lee

    Full Text Available While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral

  11. Expression analysis of the N-Myc downstream-regulated gene 1 indicates that myelinating Schwann cells are the primary disease target in hereditary motor and sensory neuropathy-Lom.

    Science.gov (United States)

    Berger, Philipp; Sirkowski, Erich E; Scherer, Steven S; Suter, Ueli

    2004-11-01

    Mutations in the gene encoding N-myc downstream-regulated gene-1 (NDRG1) lead to truncations of the encoded protein and are associated with an autosomal recessive demyelinating neuropathy--hereditary motor and sensory neuropathy-Lom. NDRG1 protein is highly expressed in peripheral nerve and is localized in the cytoplasm of myelinating Schwann cells, including the paranodes and Schmidt-Lanterman incisures. In contrast, sensory and motor neurons as well as their axons lack NDRG1. NDRG1 mRNA levels in developing and injured adult sciatic nerves parallel those of myelin-related genes, indicating that the expression of NDRG1 in myelinating Schwann cells is regulated by axonal interactions. Oligodendrocytes also express NDRG1, and the subtle CNS deficits of affected patients may result from a lack of NDRG1 in these cells. Our data predict that the loss of NDRG1 leads to a Schwann cell autonomous phenotype resulting in demyelination, with secondary axonal loss.

  12. Macrophage depletion and Schwann cell transplantation reduce cyst size after rat contusive spinal cord injury.

    Science.gov (United States)

    Lee, Yee-Shuan; Funk, Lucy H; Lee, Jae K; Bunge, Mary Bartlett

    2018-04-01

    Schwann cell transplantation is a promising therapy for the treatment of spinal cord injury (SCI) and is currently in clinical trials. In our continuing efforts to improve Schwann cell transplantation strategies, we sought to determine the combined effects of Schwann cell transplantation with macrophage depletion. Since macrophages are major inflammatory contributors to the acute spinal cord injury, and are the major phagocytic cells, we hypothesized that transplanting Schwann cells after macrophage depletion will improve cell survival and integration with host tissue after SCI. To test this hypothesis, rat models of contusive SCI at thoracic level 8 were randomly subjected to macrophage depletion or not. In rat subjected to macrophage depletion, liposomes filled with clodronate were intraperitoneally injected at 1, 3, 6, 11, and 18 days post injury. Rats not subjected to macrophage depletion were intraperitoneally injected with liposomes filled with phosphate buffered saline. Schwann cells were transplanted 1 week post injury in all rats. Biotinylated dextran amine (BDA) was injected at thoracic level 5 to evalute axon regeneration. The Basso, Beattie, and Bresnahan locomotor test, Gridwalk test, and sensory test using von Frey filaments were performed to assess functional recovery. Immunohistochemistry was used to detect glial fibrillary acidic protein, neurofilament, and green fluorescent protein (GFP), and also to visulize BDA-labelled axons. The GFP labeled Schwann cell and cyst and lesion volumes were quantified using stained slides. The numbers of BDA-positive axons were also quantified. At 8 weeks after Schwann cell transplantation, there was a significant reduction in cyst and lesion volumes in the combined treatment group compared to Schwann cell transplantation alone. These changes were not associated, however, with improved Schwann cell survival, axon growth, or locomotor recovery. Although combining Schwann cell transplantation with macrophage

  13. Macrophage depletion and Schwann cell transplantation reduce cyst size after rat contusive spinal cord injury

    Science.gov (United States)

    Lee, Yee-Shuan; Funk, Lucy H.; Lee, Jae K.; Bunge, Mary Bartlett

    2018-01-01

    Schwann cell transplantation is a promising therapy for the treatment of spinal cord injury (SCI) and is currently in clinical trials. In our continuing efforts to improve Schwann cell transplantation strategies, we sought to determine the combined effects of Schwann cell transplantation with macrophage depletion. Since macrophages are major inflammatory contributors to the acute spinal cord injury, and are the major phagocytic cells, we hypothesized that transplanting Schwann cells after macrophage depletion will improve cell survival and integration with host tissue after SCI. To test this hypothesis, rat models of contusive SCI at thoracic level 8 were randomly subjected to macrophage depletion or not. In rat subjected to macrophage depletion, liposomes filled with clodronate were intraperitoneally injected at 1, 3, 6, 11, and 18 days post injury. Rats not subjected to macrophage depletion were intraperitoneally injected with liposomes filled with phosphate buffered saline. Schwann cells were transplanted 1 week post injury in all rats. Biotinylated dextran amine (BDA) was injected at thoracic level 5 to evalute axon regeneration. The Basso, Beattie, and Bresnahan locomotor test, Gridwalk test, and sensory test using von Frey filaments were performed to assess functional recovery. Immunohistochemistry was used to detect glial fibrillary acidic protein, neurofilament, and green fluorescent protein (GFP), and also to visulize BDA-labelled axons. The GFP labeled Schwann cell and cyst and lesion volumes were quantified using stained slides. The numbers of BDA-positive axons were also quantified. At 8 weeks after Schwann cell transplantation, there was a significant reduction in cyst and lesion volumes in the combined treatment group compared to Schwann cell transplantation alone. These changes were not associated, however, with improved Schwann cell survival, axon growth, or locomotor recovery. Although combining Schwann cell transplantation with macrophage

  14. Macrophage depletion and Schwann cell transplantation reduce cyst size after rat contusive spinal cord injury

    Directory of Open Access Journals (Sweden)

    Yee-Shuan Lee

    2018-01-01

    Full Text Available Schwann cell transplantation is a promising therapy for the treatment of spinal cord injury (SCI and is currently in clinical trials. In our continuing efforts to improve Schwann cell transplantation strategies, we sought to determine the combined effects of Schwann cell transplantation with macrophage depletion. Since macrophages are major inflammatory contributors to the acute spinal cord injury, and are the major phagocytic cells, we hypothesized that transplanting Schwann cells after macrophage depletion will improve cell survival and integration with host tissue after SCI. To test this hypothesis, rat models of contusive SCI at thoracic level 8 were randomly subjected to macrophage depletion or not. In rat subjected to macrophage depletion, liposomes filled with clodronate were intraperitoneally injected at 1, 3, 6, 11, and 18 days post injury. Rats not subjected to macrophage depletion were intraperitoneally injected with liposomes filled with phosphate buffered saline. Schwann cells were transplanted 1 week post injury in all rats. Biotinylated dextran amine (BDA was injected at thoracic level 5 to evalute axon regeneration. The Basso, Beattie, and Bresnahan locomotor test, Gridwalk test, and sensory test using von Frey filaments were performed to assess functional recovery. Immunohistochemistry was used to detect glial fibrillary acidic protein, neurofilament, and green fluorescent protein (GFP, and also to visulize BDA-labelled axons. The GFP labeled Schwann cell and cyst and lesion volumes were quantified using stained slides. The numbers of BDA-positive axons were also quantified. At 8 weeks after Schwann cell transplantation, there was a significant reduction in cyst and lesion volumes in the combined treatment group compared to Schwann cell transplantation alone. These changes were not associated, however, with improved Schwann cell survival, axon growth, or locomotor recovery. Although combining Schwann cell transplantation with

  15. Schwann cells promote post-traumatic nerve inflammation and neuropathic pain through MHC class II.

    Science.gov (United States)

    Hartlehnert, Maike; Derksen, Angelika; Hagenacker, Tim; Kindermann, David; Schäfers, Maria; Pawlak, Mathias; Kieseier, Bernd C; Meyer Zu Horste, Gerd

    2017-10-02

    The activation of T helper cells requires antigens to be exposed on the surface of antigen presenting cells (APCs) via MHC class II (MHC-II) molecules. Expression of MHC-II is generally limited to professional APCs, but other cell types can express MHC-II under inflammatory conditions. However, the importance of these conditional APCs is unknown. We and others have previously shown that Schwann cells are potentially conditional APCs, but the functional relevance of MHC-II expression by Schwann cells has not been studied in vivo. Here, we conditionally deleted the MHC-II β-chain from myelinating Schwann cells in mice and investigated how this influenced post-traumatic intraneural inflammation and neuropathic pain using the chronic constriction injury (CCI) model. We demonstrate that deletion of MHC-II in myelinating Schwann cells reduces thermal hyperalgesia and, to a lesser extent, also diminishes mechanical allodynia in CCI in female mice. This was accompanied by a reduction of intraneural CD4+ T cells and greater preservation of preferentially large-caliber axons. Activation of T helper cells by MHC-II on Schwann cells thus promotes post-traumatic axonal loss and neuropathic pain. Hence, we provide experimental evidence that Schwann cells gain antigen-presenting function in vivo and modulate local immune responses and diseases in the peripheral nerves.

  16. The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells.

    Science.gov (United States)

    Jin, Song-Hyo; An, Sung-Kwan; Lee, Seong-Beom

    2017-06-01

    Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.

  17. Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Kristel Kegler

    Full Text Available Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide evidence for regeneration promoting cells recruitable for therapeutic purposes. There is evidence that neurotrophin receptor p75 (p75NTR-expressing cells emerge in the lesioned CNS. However, the phenotype and identity of these cells, and signals triggering their in situ generation under normal conditions and certain pathological situations has remained enigmatic. In the present study, we used a spontaneous, idiopathic and inflammatory CNS condition in dogs with prominent lympho-histiocytic infiltration as a model to study the phenotype of Schwann cells and their relation to Schwann cell remyelination within the CNS. Furthermore, the phenotype of p75NTR-expressing cells within the injured CNS was compared to their counter-part in control sciatic nerve and after peripheral nerve injury. In addition, organotypic slice cultures were used to further elucidate the origin of p75NTR-positive cells. In cerebral and cerebellar white and grey matter lesions as well as in the brain stem, p75NTR-positive cells co-expressed the transcription factor Sox2, but not GAP-43, GFAP, Egr2/Krox20, periaxin and PDGFR-α. Interestingly, and contrary to the findings in control sciatic nerves, p75NTR-expressing cells only co-localized with Sox2 in degenerative neuropathy, thus suggesting that such cells might represent dedifferentiated Schwann cells both in the injured CNS and PNS. Moreover, effective Schwann cell remyelination represented by periaxin- and P0-positive mature myelinating Schwann cells, was strikingly associated with the presence of p75NTR/Sox2-expressing Schwann cells. Intriguingly, the emergence of dedifferentiated Schwann cells was not affected by astrocytes, and a macrophage-dominated inflammatory response provided an adequate environment for Schwann cells plasticity within the injured CNS. Furthermore, axonal damage was reduced in brain stem areas

  18. Development of a Functional Schwann Cell Phenotype from Autologous Porcine Bone Marrow Mononuclear Cells for Nerve Repair

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    Michael J. Rutten

    2012-01-01

    Full Text Available Adult bone marrow mononuclear cells (BM-MNCs are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF expression. Addition of neuregulin (1–25 nM increased p75(NGF levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP=ATP>ADP>AMP>adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

  19. Adipose-Derived Stem Cells Promote Peripheral Nerve Regeneration In Vivo without Differentiation into Schwann-Like Lineage.

    Science.gov (United States)

    Sowa, Yoshihiro; Kishida, Tsunao; Imura, Tetsuya; Numajiri, Toshiaki; Nishino, Kenichi; Tabata, Yasuhiko; Mazda, Osam

    2016-02-01

    During recent decades, multipotent stem cells were found to reside in the adipose tissue, and these adipose-derived stem cells were shown to play beneficial roles, like those of Schwann cells, in peripheral nerve regeneration. However, it has not been well established whether adipose-derived stem cells offer beneficial effects to peripheral nerve injuries in vivo as Schwann cells do. Furthermore, the in situ survival and differentiation of adipose-derived stem cells after transplantation at the injured peripheral nerve tissue remain to be fully elucidated. Adipose-derived stem cells and Schwann cells were transplanted with gelatin hydrogel tubes at the artificially blunted sciatic nerve lesion in mice. Neuroregenerative abilities of them were comparably estimated. Cre-loxP-mediated fate tracking was performed to visualize survival in vivo of transplanted adipose-derived stem cells and to investigate whether they differentiated into Schwann linage cells at the peripheral nerve injury site. The transplantation of adipose-derived stem cells promoted regeneration of axons, formation of myelin, and restoration of denervation muscle atrophy to levels comparable to those achieved by Schwann cell transplantation. The adipose-derived stem cells survived for at least 4 weeks after transplantation without differentiating into Schwann cells. Transplanted adipose-derived stem cells did not differentiate into Schwann cells but promoted peripheral nerve regeneration at the injured site. The neuroregenerative ability was comparable to that of Schwann cells. Adipose-derived stem cells at an undifferentiated stage may be used as an alternative cell source for autologous cell therapy for patients with peripheral nerve injury.

  20. Effects of cholinergic compounds on the axon-Schwann cell relationship in the squid nerve fiber.

    Science.gov (United States)

    Villegas, J

    1975-04-01

    The effects of acetylcholine, carbamylcholine, D-tubocurarine, eserine, and alpha-bungarotoxin on the Schwann cell electrical potential of resting and stimulated squid nerve fibers were studied. Acetylcholine (10-7 M) and barbamylcholine (10-6 M) induce a prolonged hyper polarization in the Schwann cells of the unstimulated nerve fiber. In the presence of carbamylcholine (10-6 M) the behavior of the Schwann cell membrane to changes in the external potassium concentration approximates the behavior of an ideal potassium electrode. D-Tubocurarine (10-9 M) blocks the hyperpolarizing effects of nerve impulse trains and carbamylcholine (10-6 M), whereas at the same concentration eserine prolongs the Schwann cell hyperpolarizations induced by axon stimulation or by acetylcholine (10-7 M). alpha-Bungarotoxin (10-9M) also blocks the hyperpolarizing effect of nerve impulse trains and of carbamylcholine. D-Tubocurarine (10-5M) protects the Schwann cells against the irreversible action of alpha-bungarotoxin. These results show the existence of acetylcholine receptors in the Schwann cell membrane. Preliminary measurements of the binding of 125I-alpha bungarotoxin to the plasma membranes isolated from squid nerves also indicate the presence of acetylcholine receptors. These findings support the involvement of cholinergic mechanisms in the axon-Schwann cell relationship previously described.

  1. Observations on the interactions of Schwann cells and astrocytes following x irradiation of neonatal rat spinal cord

    Energy Technology Data Exchange (ETDEWEB)

    Blakemore, W F; Patterson, R C

    1975-10-01

    Myelination was inhibited in the spinal cord of three day-old rats with 2000 rads of x irradiation. Myelination subsequently occurred as a result of caudal migration of oligodendrocytes and extensive invasion of the cord by Schwann cells. Although oligodendrocytes were present in areas containing Schwann cells, astrocytes were absent. The presence of Schwann cells in the neuropil of the spinal cord did not stimulate production of basement membrane by astrocytes, so no new glial limiting membrane was formed. Evidence is presented which suggests that if astrocytes do not form a glial limiting membrane when opposed by large numbers of Schwann cells they are destroyed by the invading cells. It is suggested that the glial limiting membrane normally inhibits entry of Schwann cells into the central nervous system; if this is destroyed and not reconstituted, Schwann cells can migrate freely into the neuropil.

  2. He-Ne laser irradiation affects proliferation of cultured rat Schwann cells in a dose-dependent manner

    International Nuclear Information System (INIS)

    Breugel, H.H.F.I. van; Bar, P.R.

    1993-01-01

    Schwann cell proliferation is considered an essential part of Wallerian degeneration after nerve damage. Laminin, an important component of the extracellular matrix and produced by Schwann cells, provides a preferred substrate for outgrowing axons. To study whether low energy (He-Ne) laser irradiation may exert a positive effect on nerve regeneration through an effect on Schwann cells, its effect was evaluated in vitro. Schwann cells were isolated from sciatic nerves of 4-5-day old Wistar rats and cultures on 96-multiwell plates. The cells were irradiated by a He-Ne laser beam. At three consecutive days, starting either at day 5 or day 8, cells were irradiated each day for 0.5, 1, 2, 5 or 10 min. Both cell number and laminin production were determined for each irradiation condition within one experiment. Schwann cells that were irradiated from day 8 on were hardly affected by laser irradiation. However, the proliferation of cells that were irradiated starting on day 5 was significantly increased after 1, 2 and 5 min of daily irradiation, compared to non-irradiated control cultures. The lamin production per cell of these Schwann cells was not significantly altered. From these results we conclude that He-Ne laser irradiation can modulate proliferation of rat Schwann cells in vitro in a dose-dependent manner. (Author)

  3. Schwann cell seeded guidance tubes restore erectile function after ablation of cavernous nerves in rats.

    Science.gov (United States)

    May, F; Weidner, N; Matiasek, K; Caspers, C; Mrva, T; Vroemen, M; Henke, J; Lehmer, A; Schwaibold, H; Erhardt, W; Gänsbacher, B; Hartung, R

    2004-07-01

    Dissection of the cavernous nerves eliminates spontaneous erections. We evaluated the ability of Schwann cell seeded nerve guidance tubes to restore erections after bilateral cavernous nerve resection in rats. Sections (5 mm) of the cavernous nerve were excised bilaterally, followed by immediate bilateral microsurgical reconstruction. In 10 animals per group (20 study nerves) reconstruction was performed by genitofemoral nerve interposition, interposition of silicone tubes or interposition of silicone tubes seeded with homologous Schwann cells. As the control 10 animals (20 study nerves) underwent sham operation (positive control) and bilateral nerve ablation (without reconstruction) was performed in a further 10 (negative control). Erectile function was evaluated 3 months postoperatively by relaparotomy, electrical nerve stimulation and intracavernous pressure recording. After 3 months neurostimulation resulted in an intact erectile response in 90% (18 of 20) of Schwann cell grafts, while treatment with autologous nerves (30% or 6 of 20) or tubes only (50% or 10 of 20) was less successful (p Schwann cell grafts compared to results in the other treatment groups (p Schwann cell grafts. Schwann cell seeded guidance tubes restore erectile function after the ablation of cavernous nerves in rats and they are superior to autologous nerve grafts.

  4. The insulin-like growth factors I and II stimulate proliferation of different types of Schwann cells

    DEFF Research Database (Denmark)

    Sondell, M; Svenningsen, Åsa Fex; Kanje, M

    1997-01-01

    in combination with BrdU immunocytochemistry showed that around 93% of the proliferating cells in the nerve segments were Schwann cells. Immunostaining for BrdU and GFAP (glial fibrillary acid protein) showed that IGF-II enhanced proliferation of Schwann cells surrounding unmyelinated nerve fibres. In contrast......, truncated IGF-I promoted proliferation of Schwann cells of myelinated nerve fibres while insulin increased proliferation of both cell types....

  5. Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

    International Nuclear Information System (INIS)

    Thomas, Stacey L.; Deadwyler, Gail D.; Tang, Jun; Stubbs, Evan B.; Muir, David; Hiatt, Kelly K.; Clapp, D. Wade; De Vries, George H.

    2006-01-01

    Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells

  6. The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review.

    Science.gov (United States)

    Gravvanis, A I; Lavdas, A A; Papalois, A; Tsoutsos, D A; Matsas, R

    2007-01-01

    The importance of Schwann cells in promoting nerve regeneration across a conduit has been extensively reported in the literature, and Schwann cell motility has been acknowledged as a prerequisite for myelination of the peripheral nervous system during regeneration after injury. Review of recent literature and retrospective analysis of our studies with genetically modified Schwann Cells with increased motility in order to identify the underlying mechanism of action and outline the future trends in peripheral nerve repair. Schwann cell transduction with the pREV-retrovirus, for expression of Sialyl-Transferase-X, resulting in conferring Polysialyl-residues (PSA) on NCAM, increases their motility in-vitro and ensures nerve regeneration through silicone tubes after end-to-side neurorraphy in the rat sciatic nerve model, thus significantly promoting fiber maturation and functional outcome. An artificial nerve graft consisting of a type I collagen tube lined with the genetically modified Schwann cells with increased motility, used to bridge a defect in end-to-end fashion in the rat sciatic nerve model, was shown to promote nerve regeneration to a level equal to that of a nerve autograft. The use of genetically engineered Schwann cells with enhanced motility for grafting endoneural tubes promotes axonal regeneration, by virtue of the interaction of the transplanted cells with regenerating axonal growth cones as well as via the recruitment of endogenous Schwann cells. It is envisaged that mixed populations of Schwann cells, expressing PSA and one or more trophic factors, might further enhance the regenerating and remyelinating potential of the lesioned nerves.

  7. Fabrication of Aligned Carbon Nanotube/Polycaprolactone/Gelatin Nanofibrous Matrices for Schwann Cell Immobilization

    Directory of Open Access Journals (Sweden)

    Shiao-Wen Tsai

    2014-01-01

    Full Text Available In this study, we utilized a mandrel rotating collector consisting of two parallel, electrically conductive pieces of tape to fabricate aligned electrospun polycaprolactone/gelatin (PG and carbon nanotube/polycaprolactone/gelatin (PGC nanofibrous matrices. Furthermore, we examined the biological performance of the PGC nanofibrous and film matrices using an in vitro culture of RT4-D6P2T rat Schwann cells. Using cell adhesion tests, we found that carbon nanotube inhibited Schwann cell attachment on PGC nanofibrous and film matrices. However, the proliferation rates of Schwann cells were higher when they were immobilized on PGC nanofibrous matrices compared to PGC film matrices. Using western blot analysis, we found that NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PG nanofibrous matrices. However, the carbon nanotube inhibited NRG1 and P0 protein expression in cells immobilized on PGC film matrices. Moreover, the NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PGC film matrices. We found that the matrix topography and composition influenced Schwann cell behavior.

  8. Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation*

    Science.gov (United States)

    Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-01-01

    Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. PMID:25998127

  9. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

    Science.gov (United States)

    Jaegle, Martine; Ghazvini, Mehrnaz; Mandemakers, Wim; Piirsoo, Marko; Driegen, Siska; Levavasseur, Francoise; Raghoenath, Smiriti; Grosveld, Frank; Meijer, Dies

    2003-06-01

    The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells.

  10. Spatiotemporal distribution and function of N-cadherin in postnatal Schwann cells: A matter of adhesion?

    DEFF Research Database (Denmark)

    Corell, Mikael; Wicher, Grzegorz; Limbach, Christoph

    2010-01-01

    During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells. In this ......During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells....... In this study, we investigated the distribution of N-cadherin in the developing postnatal and adult rat peripheral nervous system. N-cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron-glial or glial-glial contacts of the sciatic nerve, dorsal root ganglia (DRG......), and myenteric plexi. In the sciatic nerve, N-cadherin decreases with age and progress of myelination. In adult animals, N-cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N-cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional...

  11. Exploration of molecular pathways mediating electric field-directed Schwann cell migration by RNA-Seq

    Science.gov (United States)

    Yao, Li; Li, Yongchao; Knapp, Jennifer; Smith, Peter

    2015-01-01

    In peripheral nervous systems, Schwann cells wrap around axons of motor and sensory neurons to form the myelin sheath. Following spinal cord injury, Schwann cells regenerate and migrate to the lesion and are involved in the spinal cord regeneration process. Transplantation of Schwann cells into injured neural tissue results in enhanced spinal axonal regeneration. Effective directional migration of Schwann cells is critical in the neural regeneration process. In this study, we report that Schwann cells migrate anodally in an applied electric field (EF). The directedness and displacement of anodal migration increased significantly when the strength of the EF increased from 50 mV/mm to 200 mV/mm. The EF did not significantly affect the cell migration speed. To explore the genes and signaling pathways that regulate cell migration in EFs, we performed a comparative analysis of differential gene expression between cells stimulated with an EF (100 mV/mm) and those without using next-generation RNA sequencing, verified by RT-qPCR. Based on the cut-off criteria (FC > 1.2, q cells versus EF-stimulated cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that compared to the control group, 21 pathways are down-regulated, while 10 pathways are up-regulated. Differentially expressed genes participate in multiple cellular signaling pathways involved in the regulation of cell migration, including pathways of regulation of actin cytoskeleton, focal adhesion, and PI3K-Akt. PMID:25557037

  12. Evidence that glutamate mediates axon-to-Schwann cell signaling in the squid.

    Science.gov (United States)

    Lieberman, E M; Abbott, N J; Hassan, S

    1989-01-01

    High-frequency stimulation (100 Hz) of isolated giant axons of the small squid Alloteuthis subulata and the large squid Loligo forbesi caused the periaxonal Schwann cell resting potential (Em = -40 mV) to hyperpolarize up to 11 mV in direct proportion to train duration and action potential amplitude. In both species, the Schwann cell also hyperpolarized up to 17 mV with the application of L-glutamate (10(-9) to 10(-6) M), in a dose-dependent manner. By contrast, in the presence of 10(-8) M d-tubocurarine (d-TC) to block the cholinergic component of the Schwann cell response, Schwann cells depolarized 8-9 mV during electrical stimulation of the axon or application of L-glutamate. In the presence of 10(-5) M 2-amino-4-phosphonobutyrate (2-APB), the hyperpolarization to glutamate and to axon stimulation was blocked, whereas the cholinergic (carbachol-induced) hyperpolarization was unaffected. In experiments with Alloteuthis, L-aspartate (10(-7) M) also caused a Schwann cell hyperpolarization, but this was not blocked by 2-APB. In tests with glutamate receptor agonists and antagonists, quisqualate (10(-5) M) produced a hyperpolarization blocked by 10(-4) M L-glutamic acid diethylester (GDEE), which also blocked the response to axonal stimulation. Kainic acid (10(-4) M) also caused a hyperpolarization, but n-methyl-D-aspartate (NMDA; 10(-4) M), ibotenate (10(-5) M), alpha-amino-3-hydroxy-5-methyl-isoxazole proprionate (AMPA; (10(-4) M), and isethionate (10(-5) M) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation.

    Science.gov (United States)

    Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-07-10

    Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Age-Dependent Schwann Cell Phenotype Regulation Following Peripheral Nerve Injury.

    Science.gov (United States)

    Chen, Wayne A; Luo, T David; Barnwell, Jonathan C; Smith, Thomas L; Li, Zhongyu

    2017-12-01

    Schwann cells are integral to the regenerative capacity of the peripheral nervous system, which declines after adolescence. The mechanisms underlying this decline are poorly understood. This study sought to compare the protein expression of Notch, c-Jun, and Krox-20 after nerve crush injury in adolescent and young adult rats. We hypothesized that these Schwann cell myelinating regulatory factors are down-regulated after nerve injury in an age-dependent fashion. Adolescent (2 months old) and young adult (12 months old) rats (n = 48) underwent sciatic nerve crush injury. Protein expression of Notch, c-Jun, and Krox-20 was quantified by Western blot analysis at 1, 3, and 7 days post-injury. Functional recovery was assessed in a separate group of animals (n = 8) by gait analysis (sciatic functional index) and electromyography (compound motor action potential) over an 8-week post-injury period. Young adult rats demonstrated a trend of delayed onset of the dedifferentiating regulatory factors, Notch and c-Jun, corresponding to the delayed functional recovery observed in young adult rats compared to adolescent rats. Compound motor action potential area was significantly greater in adolescent rats relative to young adult rats, while amplitude and velocity trended toward statistical significance. The process of Schwann cell dedifferentiation following peripheral nerve injury shows different trends with age. These trends of delayed onset of key regulatory factors responsible for Schwann cell myelination may be one of many possible factors mediating the significant differences in functional recovery between adolescent and young adult rats following peripheral nerve injury.

  15. Pro-neurogenic effects of andrographolide on RSC96 Schwann cells in vitro

    Science.gov (United States)

    Xu, Fuben; Wu, Huayu; Zhang, Kun; Lv, Peizhen; Zheng, Li; Zhao, Jinmin

    2016-01-01

    Nerve regeneration remains a challenge to the treatment of peripheral nerve injury. Andrographolide (Andro) is the main active constituent of Andrographis paniculata, which has been applied in the treatment of several diseases, including inflammation, in ancient China. Andro has been reported to facilitate the reduction of edema and to exert analgesic effects in the treatment of various diseases. These findings suggest that Andro may be considered a promising anti-inflammatory agent that may suppress destruction and accelerate proliferation of Schwann cells following peripheral nerve injury. In the present study, the effects of Andro on RSC96 cells were investigated in vitro. The RSC96 cell line is a spontaneously immortalized rat Schwann cell line, which was originally derived from a long-term culture of rat primary Schwann cells. RSC96 cells were treated with a range of 0 to 50 µM Andro prior to the MTT assay. Cell proliferation, morphology, synthesis and nerve-specific gene expression were performed to detect the effect of Andro on RSC96 cells. The results of the present study demonstrated that the recommended doses of Andro ranged between 0.78 and 12.5 µM, among which the most obvious response was observed when used at 3.125 µM (P<0.05). DNA content was improved in Andro groups compared with the control group (P<0.05). In addition, Andro was able to promote the gene expression of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and the specific Schwann cell marker S100β (P<0.05). The results of a viability assay, hematoxylin-eosin staining, and immunohistochemistry were also improved in Andro groups. These results indicated that Andro may accelerate proliferation of RSC96 cells in vitro, whilst maintaining the Schwann cell phenotype; therefore, the present study may provide valuable evidence for the further exploration of the effects of Andro on peripheral nerves. PMID:27599453

  16. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    It has been suggested that the BMSCs have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. In this study, we showed that BMSCs can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells by Brdu pulse label technology.

  17. ATP secretion from nerve trunks and Schwann cells mediated by glutamate.

    Science.gov (United States)

    Liu, Guo Jun; Bennett, Max R

    2003-11-14

    ATP release from rat sciatic nerves and from cultured Schwann cells isolated from the nerves was investigated using an online bioluminescence technique. ATP was released in relatively large amounts from rat sciatic nerve trunks during electrical stimulation. This release was blocked by the sodium channel inhibitor tetrodotoxin and the non-NMDA glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Schwann cells isolated from the nerve trunks did not release ATP when electrically stimulated but did in response to glutamate in a concentration-dependent manner. Glutamate-stimulated ATP release was inhibited by specific non-competitive AMPA receptor antagonist GYKI 52466 and competitive non-NMDA receptor antagonist CNQX. Glutamate-stimulated ATP release was decreased by inhibition of anion transporter inhibitors by furosemide, cystic fibrosis transmembrane conductance regulator by glibenclamide and exocytosis by botulinum toxin A, indicating that anion transporters and exocytosis provide the main secretion mechanisms for ATP release from the Schwann cells.

  18. Mycobacterium leprae induces NF-κB-dependent transcription repression in human Schwann cells

    International Nuclear Information System (INIS)

    Pereira, Renata M.S.; Calegari-Silva, Teresa Cristina; Hernandez, Maristela O.; Saliba, Alessandra M.; Redner, Paulo; Pessolani, Maria Cristina V.; Sarno, Euzenir N.; Sampaio, Elizabeth P.; Lopes, Ulisses G.

    2005-01-01

    Mycobacterium leprae, the causative agent of leprosy, invades peripheral nerve Schwann cells, resulting in deformities associated with this disease. NF-κB is an important transcription factor involved in the regulation of host immune antimicrobial responses. We aimed in this work to investigate NF-κB signaling pathways in the human ST88-14 Schwannoma cell line infected with M. leprae. Gel shift and supershift assays indicate that two NF-κB dimers, p65/p50 and p50/p50, translocate to the nucleus in Schwann cells treated with lethally irradiated M. leprae. Consistent with p65/p50 and p50/p50 activation, we observed IκB-α degradation and reduction of p105 levels. The nuclear translocation of p50/p50 complex due to M. leprae treatment correlated with repression of NF-κB-driven transcription induced by TNF-α. Moreover, thalidomide inhibited p50 homodimer nuclear translocation induced by M. leprae and consequently rescues Schwann cells from NF-κB-dependent transcriptional repression. Here, we report for the first time that M. leprae induces NF-κB activation in Schwann cells and thalidomide is able to modulate this activation

  19. Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae*

    Science.gov (United States)

    Medeiros, Rychelle Clayde Affonso; Girardi, Karina do Carmo de Vasconcelos; Cardoso, Fernanda Karlla Luz; Mietto, Bruno de Siqueira; Pinto, Thiago Gomes de Toledo; Gomez, Lilian Sales; Rodrigues, Luciana Silva; Gandini, Mariana; Amaral, Julio Jablonski; Antunes, Sérgio Luiz Gomes; Corte-Real, Suzana; Rosa, Patricia Sammarco; Pessolani, Maria Cristina Vidal; Nery, José Augusto da Costa; Sarno, Euzenir Nunes; Batista-Silva, Leonardo Ribeiro; Sola-Penna, Mauro; Oliveira, Marcus Fernandes; Moraes, Milton Ozório; Lara, Flavio Alves

    2016-01-01

    Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed. PMID:27555322

  20. MAL Overexpression Leads to Disturbed Expression of Genes That Influence Cytoskeletal Organization and Differentiation of Schwann Cells

    Directory of Open Access Journals (Sweden)

    Daniela Schmid

    2014-09-01

    Full Text Available In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0 and the low-affinity neurotrophin receptor p75NTR . This study shows that MAL overexpression leads to a significant reduction of Mpz and p75NTR expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination.

  1. Neurite outgrowth is significantly increased by the simultaneous presentation of Schwann cells and moderate exogenous electric fields

    Science.gov (United States)

    Koppes, Abigail N.; Seggio, Angela M.; Thompson, Deanna M.

    2011-08-01

    Axonal extension is influenced by a variety of external guidance cues; therefore, the development and optimization of a multi-faceted approach is probably necessary to address the intricacy of functional regeneration following nerve injury. In this study, primary dissociated neonatal rat dorsal root ganglia neurons and Schwann cells were examined in response to an 8 h dc electrical stimulation (0-100 mV mm-1). Stimulated samples were then fixed immediately, immunostained, imaged and analyzed to determine Schwann cell orientation and characterize neurite outgrowth relative to electric field strength and direction. Results indicate that Schwann cells are viable following electrical stimulation with 10-100 mV mm-1, and retain a normal morphology relative to unstimulated cells; however, no directional bias is observed. Neurite outgrowth was significantly enhanced by twofold following exposure to either a 50 mV mm-1 electric field (EF) or co-culture with unstimulated Schwann cells by comparison to neurons cultured alone. Neurite outgrowth was further increased in the presence of simultaneously applied cues (Schwann cells + 50 mV mm-1 dc EF), exhibiting a 3.2-fold increase over unstimulated control neurons, and a 1.2-fold increase over either neurons cultured with unstimulated Schwann cells or the electrical stimulus alone. These results indicate that dc electric stimulation in combination with Schwann cells may provide synergistic guidance cues for improved axonal growth relevant to nerve injuries in the peripheral nervous system.

  2. Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

    Directory of Open Access Journals (Sweden)

    Matthew P. Krause

    2014-07-01

    Full Text Available Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs, a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

  3. The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells

    OpenAIRE

    Jin, Song-Hyo; An, Sung-Kwan; Lee, Seong-Beom

    2017-01-01

    Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidate...

  4. The Pseudopod System for Axon-Glia Interactions: Stimulation and Isolation of Schwann Cell Protrusions that Form in Response to Axonal Membranes.

    Science.gov (United States)

    Poitelon, Yannick; Feltri, M Laura

    2018-01-01

    In the peripheral nervous system, axons dictate the differentiation state of Schwann cells. Most of this axonal influence on Schwann cells is due to juxtacrine interactions between axonal transmembrane molecules (e.g., the neuregulin growth factor) and receptors on the Schwann cell (e.g., the ErbB2/ErbB3 receptor). The fleeting nature of this interaction together with the lack of synchronicity in the development of the Schwann cell population limits our capability to study this phenomenon in vivo. Here we present a simple Boyden Chamber-based method to study this important cell-cell interaction event. We isolate the early protrusions of Schwann cells that are generated in response to juxtacrine stimulation by sensory neuronal membranes. This method is compatible with a large array of current biochemical analyses and provides an effective approach to study biomolecules that are differentially localized in Schwann cell protrusions and cell bodies in response to axonal signals. A similar approach can be extended to different kinds of cell-cell interactions.

  5. Electric field stimulation through a substrate influences Schwann cell and extracellular matrix structure

    Science.gov (United States)

    Nguyen, Hieu T.; Wei, Claudia; Chow, Jacqueline K.; Nguy, Lindsey; Nguyen, Hieu K.; Schmidt, Christine E.

    2013-08-01

    Objective. Electric field (EF) stimulation has been used to cue cell growth for tissue engineering applications. In this study, we explore the electrical parameters and extracellular mechanisms that elicit changes in cell behavior when stimulated through the substrate. Approach. Rat Schwann cell morphology was compared when exposed to EF through the media or a conductive indium tin oxide substrate. Ionic and structural effects were then analyzed on Matrigel and collagen I, respectively. Main results. When stimulating through media, cells had greater alignment perpendicular to the EF with higher current densities (106 mA cm-2 at 245 mV mm-1), and reached maximum alignment within 8 h. Stimulation through the substrate with EF (up to 110 mV mm-1) did not affect Schwann cell orientation, however the EF caused extracellular matrix (ECM) coatings on substrates to peel away, suggesting EF can physically change the ECM. Applying alternating current (ac) 2-1000 Hz signals through the media or substrate both caused cells to flatten and protrude many processes, without preferential alignment. Matrigel exposed to a substrate EF of 10 mV mm-1 for 2 h had a greater calcium concentration near the cathode, but quickly dissipated when the EF was removed. Schwann cells seeded 7 d after gels were exposed to substrate EF still aligned perpendicular to the EF direction. Microscopy of collagen I exposed to substrate EF shows alignment and bundling of fibrils. Significance. These findings demonstrate EF exposure can control Schwann cell alignment and morphology, change ECM bulk/surface architecture, and align ECM structures.

  6. Mycolactone cytotoxicity in Schwann cells could explain nerve damage in Buruli ulcer.

    Directory of Open Access Journals (Sweden)

    Junichiro En

    2017-08-01

    Full Text Available Buruli ulcer is a chronic painless skin disease caused by Mycobacterium ulcerans. The local nerve damage induced by M. ulcerans invasion is similar to the nerve damage evoked by the injection of mycolactone in a Buruli ulcer mouse model. In order to elucidate the mechanism of this nerve damage, we tested and compared the cytotoxic effect of synthetic mycolactone A/B on cultured Schwann cells, fibroblasts and macrophages. Mycolactone induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. These results suggest that mycolactone is a key substance in the production of nerve damage of Buruli ulcer.

  7. Neural stem cells promote nerve regeneration through IL12-induced Schwann cell differentiation.

    Science.gov (United States)

    Lee, Don-Ching; Chen, Jong-Hang; Hsu, Tai-Yu; Chang, Li-Hsun; Chang, Hsu; Chi, Ya-Hui; Chiu, Ing-Ming

    2017-03-01

    Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6-fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5-fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Effects of Schwann cell alignment along the oriented electrospun chitosan nanofibers on nerve regeneration.

    Science.gov (United States)

    Wang, Wei; Itoh, Soichiro; Konno, Katsumi; Kikkawa, Takeshi; Ichinose, Shizuko; Sakai, Katsuyoshi; Ohkuma, Tsuneo; Watabe, Kazuhiko

    2009-12-15

    We have constructed a chitosan nonwoven nanofiber mesh tube consisting of oriented fibers by the electrospinning method. The efficacy of oriented nanofibers on Schwann cell alignment and positive effect of this tube on peripheral nerve regeneration were confirmed. The physical properties of the chitosan nanofiber mesh sheets prepared by electrospinning with or without fiber orientation were characterized. Then, immortalized Schwann cells were cultured on these sheets. Furthermore, the chitosan nanofiber mesh tubes with or without orientation, and bilayered chitosan mesh tube with an inner layer of oriented nanofibers and an outer layer of randomized nanofibers were bridgegrafted into rat sciatic nerve defect. As a result of fiber orientation, the tensile strength along the axis of the sheet increased. Because Schwann cells aligned along the nanofibers, oriented fibrous sheets could exhibit a Schwann cell column. Functional recovery and electrophysiological recovery occurred in time in the oriented group as well as in the bilayered group, and approximately matched those in the isograft. Furthermore, histological analysis revealed that the sprouting of myelinated axons occurred vigorously followed by axonal maturation in the isograft, oriented, and bilayered group in the order. The oriented chitosan nanofiber mesh tube may be a promising substitute for autogenous nerve graft.

  9. The influence of electrospun fibre size on Schwann cell behaviour and axonal outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Gnavi, S., E-mail: sara.gnavi@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Fornasari, B.E., E-mail: benedettaelena.fornasari@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Tonda-Turo, C., E-mail: chiara.tondaturo@polito.it [Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Politecnico of Torino, Torino 10100 (Italy); Ciardelli, G., E-mail: gianluca.ciardelli@polito.it [Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Politecnico of Torino, Torino 10100 (Italy); CNR-IPCF UOS, Pisa 56124 (Italy); Zanetti, M., E-mail: marco.zanetti@unito.it [Nanostructured Interfaces and Surfaces, Department of Chemistry, University of Torino, Torino 10100 (Italy); Geuna, S., E-mail: stefano.geuna@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Perroteau, I., E-mail: isabelle.perroteau@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy)

    2015-03-01

    Fibrous substrates functioning as temporary extracellular matrices can be prepared easily by electrospinning, yielding fibrous matrices suitable as internal fillers for nerve guidance channels. In this study, gelatin micro- or nano-fibres were prepared by electrospinning by tuning the gelatin concentration and solution flow rate. The effect of gelatin fibre diameter on cell adhesion and proliferation was tested in vitro using explant cultures of Schwann cells (SC) and dorsal root ganglia (DRG). Cell adhesion was assessed by quantifying the cell spreading area, actin cytoskeleton organization and focal adhesion complex formation. Nano-fibres promoted cell spreading and actin cytoskeleton organization, increasing cellular adhesion and the proliferation rate. However, both migration rate and motility, quantified by transwell and time lapse assays respectively, were greater in cells cultured on micro-fibres. Finally, there was more DRG axon outgrowth on micro-fibres. These data suggest that the topography of electrospun gelatin fibres can be adjusted to modulate SC and axon organization and that both nano- and micro-fibres are promising fillers for the design of devices for peripheral nerve repair. - Highlights: • Electrospinning used to produce gelatin nano- and micro-fibre matrices. • Nano-fibre matrices promote Schwann cell organization and increase proliferation rate. • Micro-fibre matrices promote Schwann cell migration. • Micro-fibre matrices promote axonal outgrowth.

  10. Schwann Cells Metabolize Extracellular 2′,3′-cAMP to 2′-AMP

    Science.gov (United States)

    Verrier, Jonathan D.; Kochanek, Patrick M.

    2015-01-01

    The 3′,5′-cAMP–adenosine pathway (3′,5′-cAMP→5′-AMP→adenosine) and the 2′,3′-cAMP–adenosine pathway (2′,3′-cAMP→2′-AMP/3′-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2′,3′-cAMP–adenosine pathway via their robust expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; converts 2′,3′-cAMP to 2′-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2′,3′-cAMP–adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2′,3′-cAMP–adenosine pathway to the 3′,5′-cAMP–adenosine pathway in Schwann cells, we examined the metabolism of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in primary rat Schwann cells in culture. Addition of 2′,3′-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2′-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2′,3′-cAMP to 3′-AMP or 3′,5′-cAMP to either 3′-AMP or 5′-AMP. Although Schwann cells slightly converted 2′,3′-cAMP and 2′-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2′,3′-cAMP and extracellular 2′-AMP. These findings indicate that Schwann cells do not have a robust 3′,5′-cAMP–adenosine pathway but do have a 2′,3′-cAMP–adenosine pathway; however, because the pathway mostly involves 2′-AMP formation rather than 3′-AMP, and because the conversion of 2′-AMP to adenosine is slow, metabolism of 2′,3′-cAMP mostly results in the accumulation of 2′-AMP. Accumulation of 2′-AMP in peripheral nerves postinjury could have

  11. Schwann cell interactions with polymer films are affected by groove geometry and film hydrophilicity

    International Nuclear Information System (INIS)

    Mobasseri, S A; Downes, S; Terenghi, G

    2014-01-01

    We have developed a biodegradable polymer scaffold made of a polycaprolactone/polylactic acid (PCL/PLA) film. Surface properties such as topography and chemistry have a vital influence on cell–material interactions. Surface modifications of PCL/PLA films were performed using topographical cues and UV–ozone treatment to improve Schwann cell organisation and behaviour. Schwann cell attachment, alignment and proliferation were evaluated on the grooved UV–ozone treated and non-treated films. Solvent casting of the polymer solution on patterned silicon substrates resulted in films with different groove shapes: V (V), sloped (SL) and square (SQ) shapes. Pitted films, with no grooves, were prepared as a negative control. The UV–ozone treatment was performed to increase hydrophilicity. The process specifications for UV–ozone treatment were evaluated and 5 min radiation time and 6 cm distance to the UV source were suggested as the optimal practise. When cultured on grooved films, Schwann cells elongated on the V and SL shape grooves without crossing over, and grew in the direction of the grooves. However, there was less elongation with more crossing over on the SQ shape grooves. The maximum cell length (511 μm) was observed on the treated V-grooved films. The cells cultured on pitted UV–ozone treated surfaces showed random arrangements with no increase in length. We have demonstrated that the synergic effects of physical cues combined with UV–ozone treatment have the potential to enhance Schwann cell morphology and alignment. (paper)

  12. Cellulose/soy protein isolate composite membranes: evaluations of in vitro cytocompatibility with Schwann cells and in vivo toxicity to animals.

    Science.gov (United States)

    Luo, Lihua; Gong, Wenrong; Zhou, Yi; Yang, Lin; Li, Daokun; Huselstein, Celine; Wang, Xiong; He, Xiaohua; Li, Yinping; Chen, Yun

    2015-01-01

    To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals. A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard. The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals. The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

  13. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration.

    Science.gov (United States)

    Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Peng, Shih-Jung; Tang, Shiue-Cheng

    2015-02-01

    The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  14. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    Directory of Open Access Journals (Sweden)

    Jyuhn-Huarng Juang

    2015-02-01

    Full Text Available The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  15. Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability.

    Science.gov (United States)

    Kraus, Armin; Täger, Joachim; Kohler, Konrad; Haerle, Max; Werdin, Frank; Schaller, Hans-Eberhard; Sinis, Nektarios

    2010-11-01

    To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC). The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined. Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-L-ornithine coated plates was compromised by either method. Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

  16. Interactions between intraspinal Schwann cells and the cellular constituents normally occurring in the spinal cord: an ultrastructural study in the irradiated rat

    International Nuclear Information System (INIS)

    Sims, T.J.; Gilmore, S.A.

    1983-01-01

    Relationships between intraspinal Schwann cells and neuroglia, particularly astrocytes, were studied following X-irradiation of the spinal cord in 3-day old rats. Initially, this exposure results in a depletion of the neuroglial population. By 10 days post-irradiation (P-I), gaps occur in the glia limitans, although the overlying basal lamina remains intact. Development of and myelination by intraspinal Schwann cells is well underway by 15 days P-I. These Schwann cell-occupied regions have a paucity of astrocyte processes, a finding which persists throughout the study (60 days P-I), and several types of Schwann cell-neuroglial interfaces are observed. The gaps in the glia limitans widen as the P-I interval increases. At 45 and 60 days P-I, the basal lamina no longer forms a singular, continuous covering over the spinal cord surface, but follows instead a rather tortuous course over the disrupted glia limitans and the intraspinal Schwann cells. Although the mode of initial occurrence of Schwann cells within the spinal cord is not yet understood, the data indicate that the astrocyte population is involved in that process, as well as in limiting the further development of Schwann cells within the substance of the spinal cord. (Auth.)

  17. Graded Elevation of c-Jun in Schwann Cells In Vivo: Gene Dosage Determines Effects on Development, Remyelination, Tumorigenesis, and Hypomyelination.

    Science.gov (United States)

    Fazal, Shaline V; Gomez-Sanchez, Jose A; Wagstaff, Laura J; Musner, Nicolo; Otto, Georg; Janz, Martin; Mirsky, Rhona; Jessen, Kristján R

    2017-12-13

    Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated ∼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19 ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury. SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis

  18. Allotransplanted DRG neurons or Schwann cells affect functional recovery in a rodent model of sciatic nerve injury.

    Science.gov (United States)

    Dayawansa, Samantha; Wang, Ernest W; Liu, Weimin; Markman, John D; Gelbard, Harris A; Huang, Jason H

    2014-11-01

    In this study, the functional recoveries of Sprague-Dawley rats following repair of a complete sciatic nerve transection using allotransplanted dorsal root ganglion (DRG) neurons or Schwann cells were examined using a number of outcome measures. Four groups were compared: (1) repair with a nerve guide conduit seeded with allotransplanted Schwann cells harvested from Wistar rats, (2) repair with a nerve guide conduit seeded with DRG neurons, (3) repair with solely a nerve guide conduit, and (4) sham-surgery animals where the sciatic nerve was left intact. The results corroborated our previous reported histology findings and measures of immunogenicity. The Wistar-DRG-treated group achieved the best recovery, significantly outperforming both the Wistar-Schwann group and the nerve guide conduit group in the Von Frey assay of touch response (P DRG and Wistar-Schwann seeded repairs showed lower frequency and severity in an autotomy measure of the self-mutilation of the injured leg because of neuralgia. These results suggest that in complete peripheral nerve transections, surgical repair using nerve guide conduits with allotransplanted DRG and Schwann cells may improve recovery, especially DRG neurons, which elicit less of an immune response.

  19. Schwann cell-mediated delivery of glial cell line-derived neurotrophic factor restores erectile function after cavernous nerve injury.

    Science.gov (United States)

    May, Florian; Buchner, Alexander; Schlenker, Boris; Gratzke, Christian; Arndt, Christian; Stief, Christian; Weidner, Norbert; Matiasek, Kaspar

    2013-03-01

    To evaluate the time-course of functional recovery after cavernous nerve injury using glial cell line-derived neurotrophic factor-transduced Schwann cell-seeded silicon tubes. Sections of the cavernous nerves were excised bilaterally (5 mm), followed by immediate bilateral surgical repair. A total of 20 study nerves per group were reconstructed by interposition of empty silicon tubes and silicon tubes seeded with either glial cell line-derived neurotrophic factor-overexpressing or green fluorescent protein-expressing Schwann cells. Control groups were either sham-operated or received bilateral nerve transection without nerve reconstruction. Erectile function was evaluated by relaparotomy, electrical nerve stimulation and intracavernous pressure recording after 2, 4, 6, 8 and 10 weeks. The animals underwent re-exploration only once, and were killed afterwards. The nerve grafts were investigated for the maturation state of regenerating nerve fibers and the fascular composition. Recovery of erectile function took at least 4 weeks in the current model. Glial cell line-derived neurotrophic factor-transduced Schwann cell grafts restored erectile function better than green fluorescent protein-transduced controls and unseeded conduits. Glial cell line-derived neurotrophic factor-transduced grafts promoted an intact erectile response (4/4) at 4, 6, 8 and 10 weeks that was overall significantly superior to negative controls (P cell line-derived neurotrophic factor-transduced grafts compared with negative controls (P = 0.018) and unseeded tubes (P = 0.034). Return of function was associated with the electron microscopic evidence of preganglionic myelinated nerve fibers and postganglionic unmyelinated axons. Schwann cell-mediated delivery of glial cell line-derived neurotrophic factor presents a viable approach for the treatment of erectile dysfunction after cavernous nerve injury. © 2013 The Japanese Urological Association.

  20. Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

    Science.gov (United States)

    Arthur-Farraj, Peter J; Morgan, Claire C; Adamowicz, Martyna; Gomez-Sanchez, Jose A; Fazal, Shaline V; Beucher, Anthony; Razzaghi, Bonnie; Mirsky, Rhona; Jessen, Kristjan R; Aitman, Timothy J

    2017-09-12

    Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea.

    Science.gov (United States)

    Zhang, Zhi-Jian; Guan, Hong-Xia; Yang, Kun; Xiao, Bo-Kui; Liao, Hua; Jiang, Yang; Zhou, Tao; Hua, Qing-Quan

    2017-10-01

    This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL). Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells. Ouabain at the dosages of 0.5 mM, 1 mM and 3 mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3 mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure. The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.

  2. Low-frequency electrical stimulation induces the proliferation and differentiation of peripheral blood stem cells into Schwann cells.

    Science.gov (United States)

    Gu, Xudong; Fu, Jianming; Bai, Jing; Zhang, Chengwen; Wang, Jing; Pan, Wenping

    2015-02-01

    Functional recovery after peripheral nerve injury remains a tough problem at present. Specifically, a type of glial cell exists in peripheral nerves that promotes axonal growth and myelin formation and secretes various active substances, such as neurotrophic factors, extracellular matrix and adherence factors. These substances have important significance for the survival, growth and regeneration of nerve fibers. Numerous recent studies have shown that electrical stimulation can increase the number of myelinated nerve fibers. However, whether electrical stimulation acts on neurons or Schwann cells has not been verified in vivo. This study investigates low-frequency electrical stimulation-induced proliferation and differentiation of peripheral blood stem cells into Schwann cells and explores possible mechanisms. Peripheral blood stem cells from Sprague-Dawley rats were primarily cultured. Cells in passage 3 were divided into 4 groups: a low-frequency electrical stimulation group (20 Hz, 100 μs, 3 V), a low-frequency electrical stimulation+PD98059 (blocking the extracellular signal-regulated kinase [ERK] signaling pathway) group, a PD98059 group and a control group (no treatment). After induction, the cells were characterized. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay was employed to measure the absorbance values at 570 nm in the 4 groups. A Western blot assay was used to detect the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in each group. No significant difference in cell viability was detected before induction. Peripheral blood stem cells from the 4 groups differentiated into Schwann cells. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels were highest in the low-frequency electrical stimulation group and lowest in the ERK blockage group. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels in the low-frequency electrical stimulation+ERK blockage group were lower than those in the low-frequency electrical

  3. Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair

    NARCIS (Netherlands)

    Shakhbazau, A.; Kawasoe, J.; Hoyng, S.A.; Kumar, R.; van Minnen, J.; Verhaagen, J.; Midha, R.

    2012-01-01

    Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a

  4. Sustained Expression of Negative Regulators of Myelination Protects Schwann Cells from Dysmyelination in a Charcot-Marie-Tooth 1B Mouse Model.

    Science.gov (United States)

    Florio, Francesca; Ferri, Cinzia; Scapin, Cristina; Feltri, M Laura; Wrabetz, Lawrence; D'Antonio, Maurizio

    2018-05-02

    Schwann cell differentiation and myelination in the PNS are the result of fine-tuning of positive and negative transcriptional regulators. As myelination starts, negative regulators are downregulated, whereas positive ones are upregulated. Fully differentiated Schwann cells maintain an extraordinary plasticity and can transdifferentiate into "repair" Schwann cells after nerve injury. Reactivation of negative regulators of myelination is essential to generate repair Schwann cells. Negative regulators have also been implicated in demyelinating neuropathies, although their role in disease remains elusive. Here, we used a mouse model of Charcot-Marie-Tooth neuropathy type 1B (CMT1B), the P0S63del mouse characterized by ER stress and the activation of the unfolded protein response, to show that adult Schwann cells are in a partial differentiation state because they overexpress transcription factors that are normally expressed only before myelination. We provide evidence that two of these factors, Sox2 and Id2, act as negative regulators of myelination in vivo However, their sustained expression in neuropathy is protective because ablation of Sox2 or/and Id2 from S63del mice of both sexes results in worsening of the dysmyelinating phenotype. This is accompanied by increased levels of mutant P0 expression and exacerbation of ER stress, suggesting that limited differentiation may represent a novel adaptive mechanism through which Schwann cells counter the toxic effect of a mutant terminal differentiation protein. SIGNIFICANCE STATEMENT In many neuropathies, Schwann cells express high levels of early differentiation genes, but the significance of these altered expression remained unclear. Because many of these factors may act as negative regulators of myelination, it was suggested that their misexpression could contribute to dysmyelination. Here, we show that the transcription factors Sox2 and Id2 act as negative regulators of myelination in vivo , but that their sustained

  5. Myelin repair by Schwann cells in the regenerating goldfish visual pathway: regional patterns revealed by X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Nona, S.N.; Stafford, C.A.; Cronly-Dillon, J.R. (Manchester Univ. (United Kingdom). Inst. of Science and Technology); Duncan, A. (Guy' s Hospital, London (United Kingdom). Dept. of Anatomy); Scholes, J. (University Coll., London (United Kingdom))

    1994-07-01

    In the regenerating goldfish optic nerves, Schwann cells of unknown origin reliably infiltrate the lesion site forming a band of peripheral-type myelinating tissue by 1-2 months, sharply demarcated form the adjacent new CNS myelin. To investigate this effect, we have interfered with cell proliferation by locally X-irradiating the fish visual pathway 24 h after the lesion. As assayed by immunohistochemistry and EM, irradiation retards until 6 months formation of new myelin by Schwann cells at the lesion site, and virtually abolishes oligodendrocyte myelination distally, but has little or no effect on nerve fibre regrowth. Optic nerve astrocyte processes normally fail to re-infiltrate the lesion, but re-occupy it after irradiation, suggesting that they are normally excluded by early cell proliferation at this site. Moreover, scattered myelinating Schwann cells also appear in the oligodendrocyte-depleted distal optic nerve after irradiation, although only as far as the optic tract. (Author).

  6. Toxicity to sensory neurons and Schwann cells in experimental linezolid-induced peripheral neuropathy.

    Science.gov (United States)

    Bobylev, Ilja; Maru, Helina; Joshi, Abhijeet R; Lehmann, Helmar C

    2016-03-01

    Peripheral neuropathy is a common side effect of prolonged treatment with linezolid. This study aimed to explore injurious effects of linezolid on cells of the peripheral nervous system and to establish in vivo and in vitro models of linezolid-induced peripheral neuropathy. C57BL/6 mice were treated with linezolid or vehicle over a total period of 4 weeks. Animals were monitored by weight, nerve conduction studies and behavioural tests. Neuropathic changes were assessed by morphometry on sciatic nerves and epidermal nerve fibre density in skin sections. Rodent sensory neuron and Schwann cell cultures were exposed to linezolid in vitro and assessed for mitochondrial dysfunction. Prolonged treatment with linezolid induced a mild, predominantly small sensory fibre neuropathy in vivo. Exposure of Schwann cells and sensory neurons to linezolid in vitro caused mitochondrial dysfunction primarily in neurons (and less prominently in Schwann cells). Sensory axonopathy could be partially prevented by co-administration of the Na(+)/Ca(2+) exchanger blocker KB-R7943. Clinical and pathological features of linezolid-induced peripheral neuropathy can be replicated in in vivo and in vitro models. Mitochondrial dysfunction may contribute to the axonal damage to sensory neurons that occurs after linezolid exposure. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Dicer in Schwann cells is required for myelination and axonal integrity

    DEFF Research Database (Denmark)

    Pereira, Jorge A.; Baumann, Reto; Norrmén, Camilla

    2010-01-01

    Dicer is responsible for the generation of mature micro-RNAs (miRNAs) and loading them into RNA-induced silencing complex (RISC). RISC functions as a probe that targets mRNAs leading to translational suppression and mRNA degradation. Schwann cells (SCs) in the peripheral nervous system undergo...

  8. Mechanosensory organ regeneration in zebrafish depends on a population of multipotent progenitor cells kept latent by Schwann cells.

    Science.gov (United States)

    Sánchez, Mario; Ceci, Maria Laura; Gutiérrez, Daniela; Anguita-Salinas, Consuelo; Allende, Miguel L

    2016-04-07

    Regenerating damaged tissue is a complex process, requiring progenitor cells that must be stimulated to undergo proliferation, differentiation and, often, migratory behaviors and morphological changes. Multiple cell types, both resident within the damaged tissue and recruited to the lesion site, have been shown to participate. However, the cellular and molecular mechanisms involved in the activation of progenitor cell proliferation and differentiation after injury, and their regulation by different cells types, are not fully understood. The zebrafish lateral line is a suitable system to study regeneration because most of its components are fully restored after damage. The posterior lateral line (PLL) is a mechanosensory system that develops embryonically and is initially composed of seven to eight neuromasts distributed along the trunk and tail, connected by a continuous stripe of interneuromastic cells (INCs). The INCs remain in a quiescent state owing to the presence of underlying Schwann cells. They become activated during development to form intercalary neuromasts. However, no studies have described if INCs can participate in a regenerative event, for example, after the total loss of a neuromast. We used electroablation in transgenic larvae expressing fluorescent proteins in PLL components to completely ablate single neuromasts in larvae and adult fish. This injury results in discontinuity of the INCs, Schwann cells, and the PLL nerve. In vivo imaging showed that the INCs fill the gap left after the injury and can regenerate a new neuromast in the injury zone. Further, a single INC is able to divide and form all cell types in a regenerated neuromast and, during this process, it transiently expresses the sox2 gene, a neural progenitor cell marker. We demonstrate a critical role for Schwann cells as negative regulators of INC proliferation and neuromast regeneration, and that this inhibitory property is completely dependent on active ErbB signaling. The potential

  9. Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

    Science.gov (United States)

    He, Ya; Zhang, Peng-Zhi; Sun, Dong; Mi, Wen-Juan; Zhang, Xin-Yi; Cui, Yong; Jiang, Xing-Wang; Mao, Xiao-Bo; Qiu, Jian-Hua

    2014-04-01

    Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted

  10. Dicer in Schwann cells is required for myelination and axonal integrity

    DEFF Research Database (Denmark)

    Pereira, Jorge A.; Baumann, Reto; Norrmén, Camilla

    2010-01-01

    Dicer is responsible for the generation of mature micro-RNAs (miRNAs) and loading them into RNA-induced silencing complex (RISC). RISC functions as a probe that targets mRNAs leading to translational suppression and mRNA degradation. Schwann cells (SCs) in the peripheral nervous system undergo re...

  11. Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair.

    Science.gov (United States)

    Shakhbazau, Antos; Kawasoe, Jean; Hoyng, Stefan A; Kumar, Ranjan; van Minnen, Jan; Verhaagen, Joost; Midha, Rajiv

    2012-05-01

    Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a lentiviral vector encoding NGF (NGF-SCs). Transplantation of NGF-SCs in a rat sciatic nerve transection/repair model led to significant increase of NGF levels 2weeks after injury and correspondingly to substantial improvement in axonal regeneration. Numbers of NF200, ChAT and CGRP-positive axon profiles, as well as the gastrocnemius muscle weights, were significantly higher in the NGF-Schwann cell group compared to the animals that received control SCs transduced with a lentiviral vector encoding GFP (GFP-SCs). Comparison with other models of NGF application signifies the important role of this neurotrophin during the early stages of regeneration, and supports the importance of developing combined gene and cell therapy for peripheral nerve repair. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Suspension Matrices for Improved Schwann-Cell Survival after Implantation into the Injured Rat Spinal Cord

    Science.gov (United States)

    Patel, Vivek; Joseph, Gravil; Patel, Amit; Patel, Samik; Bustin, Devin; Mawson, David; Tuesta, Luis M.; Puentes, Rocio; Ghosh, Mousumi

    2010-01-01

    Abstract Trauma to the spinal cord produces endogenously irreversible tissue and functional loss, requiring the application of therapeutic approaches to achieve meaningful restoration. Cellular strategies, in particular Schwann-cell implantation, have shown promise in overcoming many of the obstacles facing successful repair of the injured spinal cord. Here, we show that the implantation of Schwann cells as cell suspensions with in-situ gelling laminin:collagen matrices after spinal-cord contusion significantly enhances long-term cell survival but not proliferation, as well as improves graft vascularization and the degree of axonal in-growth over the standard implantation vehicle, minimal media. The use of a matrix to suspend cells prior to implantation should be an important consideration for achieving improved survival and effectiveness of cellular therapies for future clinical application. PMID:20144012

  13. A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration.

    Science.gov (United States)

    Hadlock, T; Sundback, C; Hunter, D; Cheney, M; Vacanti, J P

    2000-04-01

    Alternatives to autografts have long been sought for use in bridging neural gaps. Many entubulation materials have been studied, although with generally disappointing results in comparison with autografts. The purpose of this study was to design a more effective neural guidance conduit, to introduce Schwann cells into the conduit, and to determine regenerative capability through it in an in vivo model. A novel, fully biodegradable polymer conduit was designed and fabricated for use in peripheral nerve repair, which approximates the macro- and microarchitecture of native peripheral nerves. It comprised a series of longitudinally aligned channels, with diameters ranging from 60 to 550 microns. The lumenal surfaces promoted the adherence of Schwann cells, whose presence is known to play a key role in nerve regeneration. This unique channel architecture increased the surface area available for Schwann cell adherence up to five-fold over that available through a simple hollow conduit. The conduit was composed of a high-molecular-weight copolymer of lactic and glycolic acids (PLGA) (MW 130,000) in an 85:15 monomer ratio. A novel foam-processing technique, employing low-pressure injection molding, was used to create highly porous conduits (approximately 90% pore volume) with continuous longitudinal channels. Using this technique, conduits were constructed containing 1, 5, 16, 45, or more longitudinally aligned channels. Prior to cellular seeding of these conduits, the foams were prewet with 50% ethanol, flushed with physiologic saline, and coated with laminin solution (10 microg/mL). A Schwann cell suspension was dynamically introduced into these processed foams at a concentration of 5 X 10(5) cells/mL, using a simple bioreactor flow loop. In vivo regeneration studies were carried out in which cell-laden five-channel polymer conduits (individual channel ID 500 microm, total conduit OD 2.3 mm) were implanted across a 7-mm gap in the rat sciatic nerve (n = 4), and midgraft

  14. Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord.

    Science.gov (United States)

    Pinzon, A; Calancie, B; Oudega, M; Noga, B R

    2001-06-01

    Central nervous system axons regenerate into a Schwann cell implant placed in the transected thoracic spinal cord of an adult rat. The present study was designed to test whether these regenerated axons are capable of conducting action potentials. Following the transection and removal of a 4- to 5-mm segment of the thoracic spinal cord (T8-T9), a polymer guidance channel filled with a mixture of adult rat Schwann cells and Matrigel was grafted into a 4- to 5-mm-long gap in the transected thoracic spinal cord. The two cut ends of the spinal cord were eased into the guidance channel openings. Transected control animals received a channel containing Matrigel only. Three months after implantation, electrophysiological studies were performed. Tungsten microelectrodes were used for monopolar stimulation of regenerated axons within the Schwann cell graft. Glass microelectrodes were used to record responses in the spinal cord rostral to the stimulation site. Evoked responses to electrical stimulation of the axon cable were found in two out of nine Schwann cell-grafted animals. These responses had approximate latencies in the range of those of myelinated axons. No responses were seen in any of the Matrigel-grafted animals. Histological analysis revealed that the two cases that showed evoked potentials had the largest number of myelinated axons present in the cable. This study demonstrates that axons regenerating through Schwann cell grafts in the complete transected spinal cord can produce measurable evoked responses following electrical stimulation. Copyright 2001 Wiley-Liss, Inc.

  15. Glucose-induced metabolic memory in Schwann cells: prevention by PPAR agonists.

    Science.gov (United States)

    Kim, Esther S; Isoda, Fumiko; Kurland, Irwin; Mobbs, Charles V

    2013-09-01

    A major barrier in reversing diabetic complications is that molecular and pathologic effects of elevated glucose persist despite normalization of glucose, a phenomenon referred to as metabolic memory. In the present studies we have investigated the effects of elevated glucose on Schwann cells, which are implicated in diabetic neuropathy. Using quantitative PCR arrays for glucose and fatty acid metabolism, we have found that chronic (>8 wk) 25 mM high glucose induces a persistent increase in genes that promote glycolysis, while inhibiting those that oppose glycolysis and alternate metabolic pathways such as fatty acid metabolism, the pentose phosphate pathway, and trichloroacetic acid cycle. These sustained effects were associated with decreased peroxisome proliferator-activated receptor (PPAR)γ binding and persistently increased reactive oxygen species, cellular NADH, and altered DNA methylation. Agonists of PPARγ and PPARα prevented select effects of glucose-induced gene expression. These observations suggest that Schwann cells exhibit features of metabolic memory that may be regulated at the transcriptional level. Furthermore, targeting PPAR may prevent metabolic memory and the development of diabetic complications.

  16. Data in support on the shape of Schwann cells and sympathetic neurons onto microconically structured silicon surfaces

    Directory of Open Access Journals (Sweden)

    C. Simitzi

    2015-09-01

    Full Text Available This article contains data related to the research article entitled “Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth” in the Biomaterials journal [1]. Scanning electron microscopy (SEM analysis is performed to investigate whether Schwann cells and sympathetic neurons alter their morphology according to the underlying topography, comprising arrays of silicon microcones with anisotropic geometrical characteristics [1]. It is observed that although soma of sympathetic neurons always preserves its round shape, this is not the case for Schwann cells that become highly polarized in high roughness microconical substrates.

  17. Schwann Cell-Mediated Preservation of Vision in Retinal Degenerative Diseases via the Reduction of Oxidative Stress: A Possible Mechanism.

    Science.gov (United States)

    Mahmoudzadeh, Raziyeh; Heidari-Keshel, Saeed; Lashay, Alireza

    2016-01-01

    After injury to the central nervous system (CNS), regeneration is often inadequate, except in the case of remyelination. This remyelination capacity of the CNS is a good example of a stem/precursor cell-mediated renewal process. Schwann cells have been found to act as remyelinating agents in the peripheral nervous system (PNS), but several studies have highlighted their potential role in remyelination in the CNS too. Schwann cells are able to protect and support retinal cells by secreting growth factors such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and basic fibroblast growth factor. Retinal degenerative diseases can be highly debilitating, and they are a major concern in countries with an ageing populations. One of the leading causes of permanent loss of vision in the West is a retinal degenerative disease known as age-related macular degeneration (AMD). In the United States, nearly 1.75 million people over the age of 40 have advanced AMD, and it is estimated that this number will increase to approximately 3 million people by 2020. One of the most common pathways involved in the initiation and development of retinal diseases is the oxidative stress pathway. In patients with diabetes, Schwann cells have been shown to be able to secrete large amounts of antioxidant enzymes that protect the PNS from the oxidative stress that results from fluctuations in blood glucose levels. This antioxidant ability may be involved in the mechanism by which Schwann cells are able to promote reconstruction in the CNS, especially in individuals with retinal injuries and degenerative diseases.

  18. 17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2016-01-01

    Full Text Available Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2 on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation.

  19. 17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

    Science.gov (United States)

    Chen, Yan; Guo, Wenjie; Li, Wenjuan; Cheng, Meng; Hu, Ying; Xu, Wenming

    2016-01-01

    Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation. PMID:27872858

  20. La célula de Schwann The Schwann Cell

    OpenAIRE

    Spinel Clara; Perdomo Sandra

    2004-01-01

    Las neuronas son las células del sistema nervioso y están recubiertas y protegidas por células gliales. En el sistema nerviosos periférico las células de Schwann (CS) son la glía de los nervios. Las prolongaciones o neuritas (axón y dendrita) de los cuerpos de las neuronas son recubiertas por las CS y constituyen las fibras nerviosas. La relación íntima entre la CS y la neurita se determina durante el desarrollo embrionario. La CS es esencial en la migración correcta de las neuritas hacia su ...

  1. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Directory of Open Access Journals (Sweden)

    José R Sotelo

    Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

  2. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  3. Sam68 promotes Schwann cell proliferation by enhancing the PI3K/Akt pathway and acts on regeneration after sciatic nerve crush

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Weijie, E-mail: 459586768@qq.com; Liu, Yuxi, E-mail: 924013616@qq.com; Wang, Youhua, E-mail: wyouhua1516@163.com

    2016-05-13

    Sam68 (Src-associated in mitosis of 68 kD), a KH domain RNA-binding protein, is not only important in signaling transduction cascades, but crucial in a variety of cellular processes. Sam68 is reported to be involved in the phospoinositide3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) signaling pathways, and it is closely associated with cell proliferation, RNA metabolism, and tumor progression. However, we know little about the role of Sam68 during peripheral nervous system injury and regeneration. In this study, we investigated the expression of Sam68 and its biological significances in sciatic nerve crush. Interestingly, we found Sam68 had a co-localization with S100 (Schwann cell marker). Moreover, after crush, Sam68 had a spatiotemporal protein expression, which was in parallel with proliferation cell nuclear antigen (PCNA). In vitro, we also observed increased expression of Sam68 during the process of TNF-α-induced Schwann cell proliferation model. Besides, flow cytometry analyses, CCK-8, and EDU were all performed with the purpose of investigating the role of Sam68 in the regulation of Schwann cell proliferation. Even more importantly, we discovered that Sam68 could enhance the phosphorylation of Akt while LY294002 (a PI3K inhibitor) obviously reversed Sam68-induced cell proliferation. Finally, we detected the variance during regeneration progress through the rat walk footprint test. In summary, all these evidences demonstrated that Sam68 might participate in Schwann cell proliferation partially via PI3K/Akt pathway and also regulate regeneration after sciatic nerve crush. -- Highlights: •The dynamic changes and location of Sam68 after sciatic nerve crush. •Sam68 promoted Schwann cell proliferation via PI3K/Akt pathway. •Sam68 modulated functional recovery after sciatic nerve crush.

  4. Sam68 promotes Schwann cell proliferation by enhancing the PI3K/Akt pathway and acts on regeneration after sciatic nerve crush

    International Nuclear Information System (INIS)

    Wu, Weijie; Liu, Yuxi; Wang, Youhua

    2016-01-01

    Sam68 (Src-associated in mitosis of 68 kD), a KH domain RNA-binding protein, is not only important in signaling transduction cascades, but crucial in a variety of cellular processes. Sam68 is reported to be involved in the phospoinositide3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) signaling pathways, and it is closely associated with cell proliferation, RNA metabolism, and tumor progression. However, we know little about the role of Sam68 during peripheral nervous system injury and regeneration. In this study, we investigated the expression of Sam68 and its biological significances in sciatic nerve crush. Interestingly, we found Sam68 had a co-localization with S100 (Schwann cell marker). Moreover, after crush, Sam68 had a spatiotemporal protein expression, which was in parallel with proliferation cell nuclear antigen (PCNA). In vitro, we also observed increased expression of Sam68 during the process of TNF-α-induced Schwann cell proliferation model. Besides, flow cytometry analyses, CCK-8, and EDU were all performed with the purpose of investigating the role of Sam68 in the regulation of Schwann cell proliferation. Even more importantly, we discovered that Sam68 could enhance the phosphorylation of Akt while LY294002 (a PI3K inhibitor) obviously reversed Sam68-induced cell proliferation. Finally, we detected the variance during regeneration progress through the rat walk footprint test. In summary, all these evidences demonstrated that Sam68 might participate in Schwann cell proliferation partially via PI3K/Akt pathway and also regulate regeneration after sciatic nerve crush. -- Highlights: •The dynamic changes and location of Sam68 after sciatic nerve crush. •Sam68 promoted Schwann cell proliferation via PI3K/Akt pathway. •Sam68 modulated functional recovery after sciatic nerve crush.

  5. Impact of scaffold micro and macro architecture on Schwann cell proliferation under dynamic conditions in a rotating wall vessel bioreactor

    International Nuclear Information System (INIS)

    Valmikinathan, Chandra M.; Hoffman, John; Yu, Xiaojun

    2011-01-01

    Over the last decade tissue engineering has emerged as a powerful alternative to regenerate lost tissues owing to trauma or tumor. Evidence shows that Schwann cell containing scaffolds have improved performance in vivo as compared to scaffolds that depend on cellularization post implantation. However, owing to limited supply of cells from the patients themselves, several approaches have been taken to enhance cell proliferation rates to produce complete and uniform cellularization of scaffolds. The most common approach is the application of a bioreactor to enhance cell proliferation rate and therefore reduce the time needed to obtain sufficiently significant number of glial cells, prior to implantation. In this study, we show the application of a rotating wall bioreactor system for studying Schwann cell proliferation on nanofibrous spiral shaped scaffolds, prepared by solvent casting and salt leaching techniques. The scaffolds were fabricated from polycaprolactone (PCL), which has ideal mechanical properties and upon degradation does not produce acidic byproducts. The spiral scaffolds were coated with aligned or random nanofibers, produced by electrospinning, to provide a substrate that mimics the native extracellular matrix and the essential contact guidance cues. At the 4 day time point, an enhanced rate of cell proliferation was observed on the open structured nanofibrous spiral scaffolds in a rotating wall bioreactor, as compared to static culture conditions. However, the cell proliferation rate on the other contemporary scaffolds architectures such as the tubular and cylindrical scaffolds show reduced cell proliferation in the bioreactor as compared to static conditions, at the same time point. Moreover, the rotating wall bioreactor does not alter the orientation or the phenotype of the Schwann cells on the aligned nanofiber containing scaffolds, wherein, the cells remain aligned along the length of the scaffolds. Therefore, these open structured spiral

  6. Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1999-01-01

    The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (10...

  7. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  8. Neuronal Regulation of Schwann Cell Mitochondrial Ca2+ Signaling during Myelination

    OpenAIRE

    Daisuke Ino; Hiroshi Sagara; Junji Suzuki; Kazunori Kanemaru; Yohei Okubo; Masamitsu Iino

    2015-01-01

    Schwann cells (SCs) myelinate peripheral neurons to promote the rapid conduction of action potentials, and the process of myelination is known to be regulated by signals from axons to SCs. Given that SC mitochondria are one of the potential regulators of myelination, we investigated whether SC mitochondria are regulated by axonal signaling. Here, we show a purinergic mechanism that sends information from neurons to SC mitochondria during myelination. Our results show that electrical stimulati...

  9. Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

    Science.gov (United States)

    Canclini, Lucía; Wallrabe, Horst; Di Paolo, Andrés; Kun, Alejandra; Calliari, Aldo; Sotelo-Silveira, José Roberto; Sotelo, José Roberto

    2014-03-15

    Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Lysophospholipid Receptors Are Differentially Expressed in Rat Terminal Schwann Cells, As Revealed by a Single Cell RT-PCR and In Situ Hybridization

    International Nuclear Information System (INIS)

    Kobashi, Hiroaki; Yaoi, Takeshi; Oda, Ryo; Okajima, Seiichiro; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Fushiki, Shinji

    2006-01-01

    Terminal Schwann cells (TSCs) that cover motor neuron terminals, are known to play an important role in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, the molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. By using our previously reported method of selectively and efficiently collecting TSCs, we have analyzed the difference in expression patterns of lysophospholipid (LPL) receptor genes (LPA 1 , LPA 2 , LPA 3 , S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) between TSCs and myelinating Schwann cells (MSCs). LPL, which includes lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), is the bioactive lipid that induces a myriad of cellular responses through specific members of G-protein coupled receptors for LPA. It turned out that LPA 3 was expressed only in TSCs, whereas S1P 1 was expressed in TSCs and skeletal muscle, but not in MSCs. Other types of LPL receptor genes, including LPA 1 , S1P 2 , S1P 3 , S1P 4 , were expressed in both types of Schwann cells. None of the LPL receptor gene family showed MSCs-specific expression

  11. Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development

    DEFF Research Database (Denmark)

    Benninger, Yves; Thurnherr, Tina; Pereira, Jorge A

    2007-01-01

    During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific......During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue...

  12. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

    NARCIS (Netherlands)

    M.M. Jaegle (Martine); M. Ghazvini (Mehrnaz); W.J. Mandemakers (Wim); M. Piirsoo (Marko); S. Driegen (Siska); F. Levavasseur (Francoise); S. Raghoenath; F.G. Grosveld (Frank); D. Meijer (Daniëlle)

    2003-01-01

    textabstractThe genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during

  13. Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.

    Science.gov (United States)

    Dilwali, Sonam; Patel, Pratik B; Roberts, Daniel S; Basinsky, Gina M; Harris, Gordon J; Emerick, Kevin S; Stankovic, Konstantina M

    2014-09-01

    Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system Comportamento de oligodendrócitos e células de Schwann em modelo experimental de desmielinização tóxica do sistema nervoso central

    Directory of Open Access Journals (Sweden)

    Dominguita Lühers Graça

    2001-06-01

    Full Text Available Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS and the peripheral nervous system (PNS. Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed.Oligodendrócitos e células de Schwann realizam a produção e manutenção das bainhas de mielina, respectivamente no sistema nervoso central (SNC e periférico (SNP. As células de Schwann, à diferença dos oligodendrócitos, são capazes de invadir o SNC para remielinizar axônios desmielinizados, sempre que os astrócitos tenham sido destruídos. O brometo de etídio é uma droga gliotóxica usada para induzir desmielinização com o desaparecimento precoce de astrócitos, de modo que as células de Schwann têm liberdade para invadir o SNC. Em ratos Wistar normais, a remielinização é detectada treze dias após desmielinização; em ratos Wistar imunossuprimidos com ciclofosfamida a reparação do tecido é tardia, enquanto que em animais tratados com ciclosporina ela

  15. Dynamic Quantification of Host Schwann Cell Migration into Peripheral Nerve Allografts

    Science.gov (United States)

    Whitlock, Elizabeth L.; Myckatyn, Terence M.; Tong, Alice Y.; Yee, Andrew; Yan, Ying; Magill, Christina K.; Johnson, Philip J.; Mackinnon, Susan E.

    2010-01-01

    Host Schwann cell (SC) migration into nerve allografts is the limiting factor in the duration of immunosuppression following peripheral nerve allotransplantation, and may be affected by different immunosuppressive regimens. Our objective was to compare SC migration patterns between clinical and experimental immunosuppression regimens both over time and at the harvest endpoint. Eighty mice that express GFP under the control of the Schwann cell specific S100 promoter were engrafted with allogeneic, nonfluorescent sciatic nerve grafts. Mice received immunosuppression with either tacrolimus (FK506), or experimental T-cell triple costimulation blockade (CSB), consisting of CTLA4-immunoglobulin fusion protein, anti-CD40 monoclonal antibody, and anti-inducible costimulator monoclonal antibody. Migration of GFP-expressing host SCs into wild-type allografts was assessed in vivo every 3 weeks until 15 weeks postoperatively, and explanted allografts were evaluated for immunohistochemical staining patterns to differentiate graft from host SCs. Immunosuppression with tacrolimus exhibited a plateau of SC migration, characterized by significant early migration (< 3 weeks) followed by a constant level of host SCs in the graft (15 weeks). At the endpoint, graft fluorescence was decreased relative to surrounding host nerve, and donor SCs persisted within the graft. CSB-treated mice displayed gradually increasing migration of host SCs into the graft, without the plateau noted in tacrolimus-treated mice, and also maintained a population of donor SCs at the 15-week endpoint. SC migration patterns are affected by immunosuppressant choice, particularly in the immediate postoperative period, and the use of a single treatment of CSB may allow for gradual population of nerve allografts with host SCs. PMID:20633557

  16. Neural differentiation of adipose-derived stem cells by indirect co-culture with Schwann cells

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    Li Xiaojie

    2009-01-01

    Full Text Available To investigate whether adipose-derived stem cells (ADSCs could be subject to neural differentiation induced only by Schwann cell (SC factors, we co-cultured ADSCs and SCs in transwell culture dishes. Immunoassaying, Western blot analysis, and RT-PCR were performed (1, 3, 7, 14 d and the co-cultured ADSCs showed gene and protein expression of S-100, Nestin, and GFAP. Further, qRT-PCR disclosed relative quantitative differences in the above three gene expressions. We think ADSCs can undergo induced neural differentiation by being co-cultured with SCs, and such differentia­tions begin 1 day after co-culture, become apparent after 7 days, and thereafter remain stable till the 14th day.

  17. Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

    Science.gov (United States)

    Cheng, Elise L; Leigh, Braden; Guymon, C Allan; Hansen, Marlan R

    2016-01-01

    The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment.

  18. Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

    Science.gov (United States)

    Petrilli, Alejandra M; Garcia, Jeanine; Bott, Marga; Klingeman Plati, Stephani; Dinh, Christine T; Bracho, Olena R; Yan, Denise; Zou, Bing; Mittal, Rahul; Telischi, Fred F; Liu, Xue-Zhong; Chang, Long-Sheng; Welling, D Bradley; Copik, Alicja J; Fernández-Valle, Cristina

    2017-05-09

    Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

  19. Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation

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    Nadia eGarcía-Mateo

    2014-11-01

    Full Text Available Management of lipids, particularly signaling lipids that control neuroinflammation, is crucial for the regeneration capability of a damaged nervous system. Knowledge of pro- and anti-inflammatory signals after nervous system injury is extensive, most of them being proteins acting through well-known receptors and intracellular cascades. However, the role of lipid binding extracellular proteins able to modify the fate of lipids released after injury is not well understood.Apolipoprotein D (ApoD is an extracellular lipid binding protein of the Lipocalin family induced upon nervous system injury. Our previous study shows that axon regeneration is delayed without ApoD, and suggests its participation in early events during Wallerian degeneration. Here we demonstrate that ApoD is expressed by myelinating and non-myelinating Schwann cells and is induced early upon nerve injury. We show that ApoD, known to bind arachidonic acid (AA, also interacts with lysophosphatidylcholine (LPC in vitro. We use an in vivo model of nerve crush injury, a nerve explant injury model, and cultured macrophages exposed to purified myelin, to uncover that: (i ApoD regulates denervated Schwann cell-macrophage signaling, dampening MCP1- and Tnf-dependent macrophage recruitment and activation upon injury; (ii ApoD controls the over-expression of the phagocytosis activator Galectin-3 by infiltrated macrophages; (iii ApoD controls the basal and injury-triggered levels of LPC and AA; (iv ApoD modifies the dynamics of myelin-macrophage interaction, favoring the initiation of phagocytosis and promoting myelin degradation.Regulation of macrophage behaviour by Schwann-derived ApoD is therefore a key mechanism conditioning nerve injury resolution. These results place ApoD as a lipid binding protein controlling the signals exchanged between glia, neurons and blood-borne cells during nerve recovery after injury, and open the possibility for a therapeutic use of ApoD as a regeneration

  20. Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

    Science.gov (United States)

    Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

    The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

  1. The Composition and Organization of Cytoplasm in Prebiotic Cells

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    Jack T. Trevors

    2011-03-01

    Full Text Available This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1–0.2 µm3 (possibly less if a nanocell prior to the first cell division.

  2. In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration and Regeneration Using Sensory Neurons from Dorsal Root Ganglia.

    Science.gov (United States)

    López-Leal, Rodrigo; Diaz, Paula; Court, Felipe A

    2018-01-01

    Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

  3. A guidance channel seeded with autologous Schwann cells for repair of cauda equina injury in a primate model.

    Science.gov (United States)

    Calancie, Blair; Madsen, Parley W; Wood, Patrick; Marcillo, Alexander E; Levi, Allan D; Bunge, Richard P

    2009-01-01

    To evaluate an implantable guidance channel (GC) seeded with autologous Schwann cells to promote regeneration of transected spinal nerve root axons in a primate model. Schwann cells were obtained from sural nerve segments of monkeys (Macaca fascicularis; cynomolgus). Cells were cultured, purified, and seeded into a PAN/PVC GC. Approximately 3 weeks later, monkeys underwent laminectomy and dural opening. Nerve roots of the L4 through L7 segments were identified visually. The threshold voltage needed to elicit hindlimb muscle electromyography (EMG) after stimulation of intact nerve roots was determined. Segments of 2 or 3 nerve roots (each approximately 8-15 mm in length) were excised. The GC containing Schwann cells was implanted between the proximal and distal stumps of these nerve roots and attached to the stumps with suture. Follow-up evaluation was conducted on 3 animals, with survival times of 9 to 14 months. Upon reexposure of the implant site, subdural nerve root adhesions were noted in all 3 animals. Several of the implanted GC had collapsed and were characterized by thin strands of connective tissue attached to either end. In contrast, 3 of the 8 implanted GC were intact and had white, glossy cables entering and exiting the conduits. Electrical stimulation of the tissue cable in each of these 3 cases led to low-threshold evoked EMG responses, suggesting that muscles had been reinnervated by axons regenerating through the repair site and into the distal nerve stump. During harvesting of the GC implant, sharp transection led to spontaneous EMG in the same 3 roots showing a low threshold to electrical stimulation, whereas no EMG was seen when harvesting nerve roots with high thresholds to elicit EMG. Histology confirmed large numbers of myelinated axons at the midpoint of 2 GC judged to have reinnervated target muscles. We found a modest rate of successful regeneration and muscle reinnervation after treatment of nerve root transection with a Schwann cell

  4. Characterization and Schwann Cell Seeding of up to 15.0 cm Long Spider Silk Nerve Conduits for Reconstruction of Peripheral Nerve Defects

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    Tim Kornfeld

    2016-11-01

    Full Text Available Nerve reconstruction of extended nerve defect injuries still remains challenging with respect to therapeutic options. The gold standard in nerve surgery is the autologous nerve graft. Due to the limitation of adequate donor nerves, surgical alternatives are needed. Nerve grafts made out of either natural or artificial materials represent this alternative. Several biomaterials are being explored and preclinical and clinical applications are ongoing. Unfortunately, nerve conduits with successful enhancement of axonal regeneration for nerve defects measuring over 4.0 cm are sparse and no conduits are available for nerve defects extending to 10.0 cm. In this study, spider silk nerve conduits seeded with Schwann cells were investigated for in vitro regeneration on defects measuring 4.0 cm, 10.0 cm and 15.0 cm in length. Schwann cells (SCs were isolated, cultured and purified. Cell purity was determined by immunofluorescence. Nerve grafts were constructed out of spider silk from Nephila edulis and decellularized ovine vessels. Finally, spider silk implants were seeded with purified Schwann cells. Cell attachment was observed within the first hour. After 7 and 21 days of culture, immunofluorescence for viability and determination of Schwann cell proliferation and migration throughout the conduits was performed. Analyses revealed that SCs maintained viable (>95% throughout the conduits independent of construct length. SC proliferation on the spider silk was determined from day 7 to day 21 with a proliferation index of 49.42% arithmetically averaged over all conduits. This indicates that spider silk nerve conduits represent a favorable environment for SC attachment, proliferation and distribution over a distance of least 15.0 cm in vitro. Thus spider silk nerve implants are a highly adequate biomaterial for nerve reconstruction.

  5. Schwann Cells in Neuromuscular Junction Formation and Maintenance.

    Science.gov (United States)

    Barik, Arnab; Li, Lei; Sathyamurthy, Anupama; Xiong, Wen-Cheng; Mei, Lin

    2016-09-21

    The neuromuscular junction (NMJ) is a tripartite synapse that is formed by motor nerve terminals, postjunctional muscle membranes, and terminal Schwann cells (TSCs) that cover the nerve-muscle contact. NMJ formation requires intimate communications among the three different components. Unlike nerve-muscle interaction, which has been well characterized, less is known about the role of SCs in NMJ formation and maintenance. We show that SCs in mice lead nerve terminals to prepatterned AChRs. Ablating SCs at E8.5 (i.e., prior nerve arrival at the clusters) had little effect on aneural AChR clusters at E13.5, suggesting that SCs may not be necessary for aneural clusters. SC ablation at E12.5, a time when phrenic nerves approach muscle fibers, resulted in smaller and fewer nerve-induced AChR clusters; however, SC ablation at E15.5 reduced AChR cluster size but had no effect on cluster density, suggesting that SCs are involved in AChR cluster maturation. Miniature endplate potential amplitude, but not frequency, was reduced when SCs were ablated at E15.5, suggesting that postsynaptic alterations may occur ahead of presynaptic deficits. Finally, ablation of SCs at P30, after NMJ maturation, led to NMJ fragmentation and neuromuscular transmission deficits. Miniature endplate potential amplitude was reduced 3 d after SC ablation, but both amplitude and frequency were reduced 6 d after. Together, these results indicate that SCs are not only required for NMJ formation, but also necessary for its maintenance; and postsynaptic function and structure appeared to be more sensitive to SC ablation. Neuromuscular junctions (NMJs) are critical for survival and daily functioning. Defects in NMJ formation during development or maintenance in adulthood result in debilitating neuromuscular disorders. The role of Schwann cells (SCs) in NMJ formation and maintenance was not well understood. We genetically ablated SCs during development and after NMJ formation to investigate the consequences

  6. Side-To-Side Nerve Bridges Support Donor Axon Regeneration Into Chronically Denervated Nerves and Are Associated With Characteristic Changes in Schwann Cell Phenotype.

    Science.gov (United States)

    Hendry, J Michael; Alvarez-Veronesi, M Cecilia; Snyder-Warwick, Alison; Gordon, Tessa; Borschel, Gregory H

    2015-11-01

    Chronic denervation resulting from long nerve regeneration times and distances contributes greatly to suboptimal outcomes following nerve injuries. Recent studies showed that multiple nerve grafts inserted between an intact donor nerve and a denervated distal recipient nerve stump (termed "side-to-side nerve bridges") enhanced regeneration after delayed nerve repair. To examine the cellular aspects of axon growth across these bridges to explore the "protective" mechanism of donor axons on chronically denervated Schwann cells. In Sprague Dawley rats, 3 side-to-side nerve bridges were placed over a 10-mm distance between an intact donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) distal nerve stump. Green fluorescent protein-expressing TIB axons grew across the bridges and were counted in cross section after 4 weeks. Immunofluorescent axons and Schwann cells were imaged over a 4-month period. Denervated Schwann cells dedifferentiated to a proliferative, nonmyelinating phenotype within the bridges and the recipient denervated CP nerve stump. As donor TIB axons grew across the 3 side-to-side nerve bridges and into the denervated CP nerve, the Schwann cells redifferentiated to the myelinating phenotype. Bridge placement led to an increased mass of hind limb anterior compartment muscles after 4 months of denervation compared with muscles whose CP nerve was not "protected" by bridges. This study describes patterns of donor axon regeneration and myelination in the denervated recipient nerve stump and supports a mechanism where these donor axons sustain a proregenerative state to prevent deterioration in the face of chronic denervation.

  7. Hyperglycemia Alters the Schwann Cell Mitochondrial Proteome and Decreases Coupled Respiration in the Absence of Superoxide Production

    OpenAIRE

    Zhang, Liang; Yu, Cuijuan; Vasquez, Francisco E.; Galeva, Nadya; Onyango, Isaac; Swerdlow, Russell H.; Dobrowsky, Rick T.

    2010-01-01

    Hyperglycemia-induced mitochondrial dysfunction contributes to sensory neuron pathology in diabetic neuropathy. Although Schwann cells (SCs) also undergo substantial degeneration in diabetic neuropathy, the effect of hyperglycemia on SC mitochondrial proteome and mitochondrial function has not been examined. Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantify the temporal effect of hyperglycemia on the mitochondrial proteome of primary SCs isolated from neona...

  8. Distribution of elements in rat peripheral axons and nerve cell bodies determined by x-ray microprobe analysis

    Energy Technology Data Exchange (ETDEWEB)

    LoPachin, R.M. Jr.; Lowery, J.; Eichberg, J.; Kirkpatrick, J.B.; Cartwright, J. Jr.; Saubermann, A.J.

    1988-09-01

    X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.

  9. Proliferación y expresión de marcadores por células de Schwann de rata adulta en cultivo Schwann cells proliferation and marker expression on adult rat in culture

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    Martínez Constanza

    1999-06-01

    Full Text Available En este trabajo se evalúan diferentes técnicas para obten-ción y cultivo de células de Schwann provenientes del nervio periférico de rata adulta, de las cuales la que evi-dencia mejor respuesta es la que combina una degenera-ción walleriana durante 14 días in vitro, seguida de una disociación enzimática. La adición de mitógenos como la forskolina y extracto de pituitaria no muestra un efecto sobre estas células. Los niveles de enriquecimiento en células de Schwann, defi-nidos de acuerdo con patrones morfológicos y de expre-sión de marcadores tales como la proteína S-100 o la pro-teína acida fíbrilar glial (GFAP, son buenos (del orden de 80-88% hasta los ocho días de cultivo. La detección de bromodeoxiouridina (BrdU incorporada por células en fase S del ciclo celular, demuestra que en términos ge-nerales la tasa de incorporación de BrdU de las células guales del sistema nervioso periférico no cambia.This study evaluated some techniques for culture and growth of Schwann Cells from adult rats peripheral nerves. The best of these methods is a combination of in vitro Wallerian degeneration during 14 days, followed by an enzimatic dissociation with collagenase and dispase Mitogens like forskolin and pituitary extract do not have any effects on these cells. Enrichement of the culture (measure by morphological and inmunocitochemical criteria was about 80-88% until 8 days in culture. Stable Level of BrdU incorporation suggested that the population of cells entering S phase does not change.

  10. Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

    Science.gov (United States)

    Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

    1987-04-01

    Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

  11. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    OpenAIRE

    Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Peng, Shih-Jung; Tang, Shiue-Cheng

    2015-01-01

    The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histolo...

  12. Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells

    OpenAIRE

    Stevens, Beth; Ishibashi, Tomoko; Chen, Jiang-Fan; Fields, R. Douglas

    2004-01-01

    Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron–glia communication are not known. Recent research shows that adenosine is a neuron–glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility...

  13. Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

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    Sandra R Boiça Silva

    2010-08-01

    Full Text Available Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14 and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

  14. Neuronal activity in the hub of extrasynaptic Schwann cell-axon interactions

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    Chrysanthi eSamara

    2013-11-01

    Full Text Available The integrity and function of neurons depend on their continuous interactions with glial cells. In the peripheral nervous system glial functions are exerted by Schwann cells (SCs. SCs sense synaptic and extrasynaptic manifestations of action potential propagation and adapt their physiology to support neuronal activity. We review here existing literature data on extrasynaptic bidirectional axon-SC communication, focusing particularly on neuronal activity implications. To shed light on underlying mechanisms, we conduct a thorough analysis of microarray data from SC-rich mouse sciatic nerve at different developmental stages and in neuropathic models. We identify molecules that are potentially involved in SC detection of neuronal activity signals inducing subsequent glial responses. We further suggest that alterations in the activity-dependent axon-SC crosstalk impact on peripheral neuropathies. Together with previously reported data, these observations open new perspectives for deciphering glial mechanisms of neuronal function support.

  15. Direct Conversion of Human Fibroblasts into Schwann Cells that Facilitate Regeneration of Injured Peripheral Nerve In Vivo.

    Science.gov (United States)

    Sowa, Yoshihiro; Kishida, Tsunao; Tomita, Koichi; Yamamoto, Kenta; Numajiri, Toshiaki; Mazda, Osam

    2017-04-01

    Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC-specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin-forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207-1216. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  16. Transdifferentiation of brain-derived neurotrophic factor (BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.

    Science.gov (United States)

    Bierlein De la Rosa, Metzere; Sharma, Anup D; Mallapragada, Surya K; Sakaguchi, Donald S

    2017-11-01

    The use of genetically modified mesenchymal stem cells (MSCs) is a rapidly growing area of research targeting delivery of therapeutic factors for neuro-repair. Cells can be programmed to hypersecrete various growth/trophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to promote regenerative neurite outgrowth. In addition to genetic modifications, MSCs can be subjected to transdifferentiation protocols to generate neural cell types to physically and biologically support nerve regeneration. In this study, we have taken a novel approach by combining these two unique strategies and evaluated the impact of transdifferentiating genetically modified MSCs into a Schwann cell-like phenotype. After 8 days in transdifferentiation media, approximately 30-50% of transdifferentiated BDNF-secreting cells immunolabeled for Schwann cell markers such as S100β, S100, and p75 NTR . An enhancement was observed 20 days after inducing transdifferentiation with minimal decreases in expression levels. BDNF production was quantified by ELISA, and its biological activity tested via the PC12-TrkB cell assay. Importantly, the bioactivity of secreted BDNF was verified by the increased neurite outgrowth of PC12-TrkB cells. These findings demonstrate that not only is BDNF actively secreted by the transdifferentiated BDNF-MSCs, but also that it has the capacity to promote neurite sprouting and regeneration. Given the fact that BDNF production remained stable for over 20 days, we believe that these cells have the capacity to produce sustainable, effective, BDNF concentrations over prolonged time periods and should be tested within an in vivo system for future experiments. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. S100ß and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury S100ß e fator de crescimento de fibroblasto-2 estão presentes nas células de Schwann cultivadas e exercem ações parácrinas na lesão do nervo

    Directory of Open Access Journals (Sweden)

    Tatiana Duobles

    2008-12-01

    Full Text Available PURPOSE: The neurotrophic factor fibroblast growth factor-2 (FGF-2, bFGF and Ca++ binding protein S100ß are expressed by the Schwann cells of the peripheral nerves and by the satellite cells of the dorsal root ganglia (DRG. Recent studies have pointed out the importance of the molecules in the paracrine mechanisms related to neuronal maintenance and plasticity of lesioned motor and sensory peripheral neurons. Moreover, cultured Schwann cells have been employed experimentally in the treatment of central nervous system lesions, in special the spinal cord injury, a procedure that triggers an enhanced sensorymotor function. Those cells have been proposed to repair long gap nerve injury. METHODS: Here we used double labeling immunohistochemistry and Western blot to better characterize in vitro and in vivo the presence of the proteins in the Schwann cells and in the satellite cells of the DRG as well as their regulation in those cells after a crush of the rat sciatic nerve. RESULTS: FGF-2 and S100ß are present in the Schwann cells of the sciatic nerve and in the satellite cells of the DRG. S100ß positive satellite cells showed increased size of the axotomized DRG and possessed elevated amount of FGF-2 immunoreactivity. Reactive satellite cells with increased FGF-2 labeling formed a ring-like structure surrounding DRG neuronal cell bodies.Reactive S100ß positive Schwann cells of proximal stump of axotomized sciatic nerve also expressed higher amounts of FGF-2. CONCLUSION: Reactive peripheral glial cells synthesizing FGF-2 and S100ß may be important in wound repair and restorative events in the lesioned peripheral nerves.OBJETIVO: O fator neurotrófico fator de crescimento de fibroblastos-2 (FGF-2, bFGF e a proteína ligante de Ca++ S100ß são expressos pelas células de Schwann dos nervos e por células satélites do gânglio da raiz dorsal (GRD. Estudos recentes indicam a importância das moléculas nos mecanismos parácrinos relacionados

  18. Short-term low-frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain-derived neurotrophic factor on Schwann cell polarization.

    Science.gov (United States)

    Wan, Lidan; Xia, Rong; Ding, Wenlong

    2010-09-01

    Electrical stimulation (ES) has been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. However, the effect of ES on peripheral remyelination after nerve damage has been investigated less well, and the mechanism underlying its action remains unclear. In the present study, the crush-injured sciatic nerves in rats were subjected to 1 hr of continuous ES (20 Hz, 100 microsec, 3 V). Electron microscopy and nerve morphometry were performed to investigate the extent of regenerated nerve myelination. The expression profiles of P0, Par-3, and brain-derived neurotrophic factor (BDNF) in the injuried sciatic nerves and in the dorsal root ganglion neuron/Schwann cell cocultures were examined by Western blotting. Par-3 localization in the sciatic nerves was determined by immunohistochemistry to demonstrate Schwann cell polarization during myelination. We reported that 20-Hz ES increased the number of myelinated fibers and the thickness myelin sheath at 4 and 8 weeks postinjury. P0 level in the ES-treated groups, both in vitro and in vivo, was enhanced compared with the controls. The earlier peak of Par-3 in the ES-treated groups indicated an earlier initiation of Schwann cell myelination. Additionally, ES significantly elevated BDNF expression in nerve tissues and in cocultures. ES on the site of nerve injury potentiates axonal regrowth and myelin maturation during peripheral nerve regeneration. Furthermore, the therapeutic actions of ES on myelination are mediated via enhanced BDNF signals, which drive the promyelination effect on Schwann cells at the onset of myelination.

  19. Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

    Science.gov (United States)

    Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

    2017-01-01

    Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

  20. Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells.

    Science.gov (United States)

    Stevens, Beth; Ishibashi, Tomoko; Chen, Jiang-Fan; Fields, R Douglas

    2004-02-01

    Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron-glia communication are not known. Recent research shows that adenosine is a neuron-glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility that adenosine might have a similar function in communicating between axons and premyelinating SCs. Using a combination of pharmacological and molecular approaches, we found that mouse SCs in culture express functional adenosine receptors and ATP receptors, a far more complex array of purinergic receptors than thought previously. Adenosine, but not ATP, activates ERK/MAPK through stimulation of cAMP-linked A2(A) adenosine receptors. Both ATP and adenosine inhibit proliferation of SCs induced by platelet-derived growth factor (PDGF), via mechanisms that are partly independent. In contrast to ATP, adenosine failed to inhibit the differentiation of SCs to the O4+ stage. This indicates that, in addition to ATP, adenosine is an activity-dependent signaling molecule between axons and premyelinating Schwann cells, but that electrical activity, acting through adenosine, has opposite effects on the differentiation of myelinating glia in the PNS and CNS.

  1. G-CSF prevents caspase 3 activation in Schwann cells after sciatic nerve transection, but does not improve nerve regeneration.

    Science.gov (United States)

    Frost, Hanna K; Kodama, Akira; Ekström, Per; Dahlin, Lars B

    2016-10-15

    Exogenous granulocyte-colony stimulating factor (G-CSF) has emerged as a drug candidate for improving the outcome after peripheral nerve injuries. We raised the question if exogenous G-CSF can improve nerve regeneration following a clinically relevant model - nerve transection and repair - in healthy and diabetic rats. In short-term experiments, distance of axonal regeneration and extent of injury-induced Schwann cell death was quantified by staining for neurofilaments and cleaved caspase 3, respectively, seven days after repair. There was no difference in axonal outgrowth between G-CSF-treated and non-treated rats, regardless if healthy Wistar or diabetic Goto-Kakizaki (GK) rats were examined. However, G-CSF treatment caused a significant 13% decrease of cleaved caspase 3-positive Schwann cells at the lesion site in healthy rats, but only a trend in diabetic rats. In the distal nerve segments of healthy rats a similar trend was observed. In long-term experiments of healthy rats, regeneration outcome was evaluated at 90days after repair by presence of neurofilaments, wet weight of gastrocnemius muscle, and perception of touch (von Frey monofilament testing weekly). The presence of neurofilaments distal to the suture line was similar in G-CSF-treated and non-treated rats. The weight ratio of ipsi-over contralateral gastrocnemius muscles, and perception of touch at any time point, were likewise not affected by G-CSF treatment. In addition, the inflammatory response in short- and long-term experiments was studied by analyzing ED1 stainable macrophages in healthy rats, but in neither case was any attenuation seen at the injury site or distal to it. G-CSF can prevent caspase 3 activation in Schwann cells in the short-term, but does not detectably affect the inflammatory response, nor improve early or late axonal outgrowth or functional recovery. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Cellular Scale Anisotropic Topography Guides Schwann Cell Motility

    Science.gov (United States)

    Mitchel, Jennifer A.; Hoffman-Kim, Diane

    2011-01-01

    Directed migration of Schwann cells (SC) is critical for development and repair of the peripheral nervous system. Understanding aspects of motility specific to SC, along with SC response to engineered biomaterials, may inform strategies to enhance nerve regeneration. Rat SC were cultured on laminin-coated microgrooved poly(dimethyl siloxane) platforms that were flat or presented repeating cellular scale anisotropic topographical cues, 30 or 60 µm in width, and observed with timelapse microscopy. SC motion was directed parallel to the long axis of the topography on both the groove floor and the plateau, with accompanying differences in velocity and directional persistence in comparison to SC motion on flat substrates. In addition, feature dimension affected SC morphology, alignment, and directional persistence. Plateaus and groove floors presented distinct cues which promoted differential motility and variable interaction with the topographical features. SC on the plateau surfaces tended to have persistent interactions with the edge topography, while SC on the groove floors tended to have infrequent contact with the corners and walls. Our observations suggest the capacity of SC to be guided without continuous contact with a topographical cue. SC exhibited a range of distinct motile morphologies, characterized by their symmetry and number of extensions. Across all conditions, SC with a single extension traveled significantly faster than cells with more or no extensions. We conclude that SC motility is complex, where persistent motion requires cellular asymmetry, and that anisotropic topography with cellular scale features can direct SC motility. PMID:21949703

  3. Schwann cell response on polypyrrole substrates upon electrical stimulation.

    Science.gov (United States)

    Forciniti, Leandro; Ybarra, Jose; Zaman, Muhammad H; Schmidt, Christine E

    2014-06-01

    Current injury models suggest that Schwann cell (SC) migration and guidance are necessary for successful regeneration and synaptic reconnection after peripheral nerve injury. The ability of conducting polymers such as polypyrrole (PPy) to exhibit chemical, contact and electrical stimuli for cells has led to much interest in their use for neural conduits. Despite this interest, there has been very little research on the effect that electrical stimulation (ES) using PPy has on SC behavior. Here we investigate the mechanism by which SCs interact with PPy in the presence of an electric field. Additionally, we explored the effect that the adsorption of different serum proteins on PPy upon the application of an electric field has on SC migration. The results indicate an increase in average displacement of the SC with ES, resulting in a net anodic migration. Moreover, indirect effects of protein adsorption due to the oxidation of the film upon the application of ES were shown to have a larger effect on migration speed than on migration directionality. These results suggest that SC migration speed is governed by an integrin- or receptor-mediated mechanism, whereas SC migration directionality is governed by electrically mediated phenomena. These data will prove invaluable in optimizing conducting polymers for their different biomedical applications such as nerve repair. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study).

    Science.gov (United States)

    Hagendoorn, MJM.; Wagner, A. M.; Segers, G.; Van Der Plas, LHW.; Oostdam, A.; Van Walraven, H. S.

    1994-10-01

    In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

  5. Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

    Science.gov (United States)

    Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

    2010-08-01

    Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and

  6. Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.

    Science.gov (United States)

    Amoh, Yasuyuki; Li, Lingna; Campillo, Raul; Kawahara, Katsumasa; Katsuoka, Kensei; Penman, Sheldon; Hoffman, Robert M

    2005-12-06

    The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with beta-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine.

  7. Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits.

    Science.gov (United States)

    Das, Suprem R; Uz, Metin; Ding, Shaowei; Lentner, Matthew T; Hondred, John A; Cargill, Allison A; Sakaguchi, Donald S; Mallapragada, Surya; Claussen, Jonathan C

    2017-04-01

    Graphene-based materials (GBMs) have displayed tremendous promise for use as neurointerfacial substrates as they enable favorable adhesion, growth, proliferation, spreading, and migration of immobilized cells. This study reports the first case of the differentiation of mesenchymal stem cells (MSCs) into Schwann cell (SC)-like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC-like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance electrically stimulated/treated (etMSCs) display significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [≈85% of the etMSCs differentiated into SC-like phenotypes with ≈80 ng mL -1 of nerve growth factor (NGF) secretion vs. 75% and ≈55 ng mL -1 for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

    Directory of Open Access Journals (Sweden)

    Carolina Wählby

    2002-01-01

    Full Text Available Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.

  9. Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

    International Nuclear Information System (INIS)

    Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

    2006-01-01

    The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16 INK4a , a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis

  10. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the...

  11. Patterns of x-radiation-induced Schwann cell development in spinal cords of immature rats

    International Nuclear Information System (INIS)

    Gilmore, S.A.; Heard, J.K.; Leiting, J.E.

    1983-01-01

    Schwann cells, Schwann cell myelin, and connective tissue components develop in the spinal cord of the immature rat following exposure to x-rays. For the purposes of this paper, these intraspinal peripheral nervous tissue constituents are referred to as IPNT. A series of investigations are in progress to elucidate factors related to the development of IPNT, and the present study is a light microscopic evaluation of the relationship between the amount of radiation administered (1,000-3,000R) to the lumbosacral spinal cord in 3-day-old rats and the incidence and distribution of IPNT at intervals up to 60 days postirradiation (P-I). The results showed that IPNT was present in only 33% of the rats exposed to 1,000R, whereas its presence was observed in 86% or more of those in the 2,000-, 2,500-, and 3,000R groups. The distribution of IPNT was quite limited in the 1,000R group, where it was restricted to the spinal cord-dorsal root junction and was found in only a few sections within the irradiated area. The distribution was more widespread with increasing amounts of radiation, and IPNT occupied substantial portions of the dorsal funiculi and extended into the dorsal gray matter in the 3,000R group. In all aR mals developing IPNT in the groups receiving 2,000R or more, the IPNT was present in essentially all sections from the irradiated area. Further studies will compare in detail spinal cords exposed to 1,000R in which IPNT is an infrequent, limited occurrence with those exposed to higher doses where IPNT occurs in a more widespread fashion in essentially all animals

  12. Hierarchical thermoplastic rippled nanostructures regulate Schwann cell adhesion, morphology and spatial organization.

    Science.gov (United States)

    Masciullo, Cecilia; Dell'Anna, Rossana; Tonazzini, Ilaria; Böettger, Roman; Pepponi, Giancarlo; Cecchini, Marco

    2017-10-12

    Periodic ripples are a variety of anisotropic nanostructures that can be realized by ion beam irradiation on a wide range of solid surfaces. Only a few authors have investigated these surfaces for tuning the response of biological systems, probably because it is challenging to directly produce them in materials that well sustain long-term cellular cultures. Here, hierarchical rippled nanotopographies with a lateral periodicity of ∼300 nm are produced from a gold-irradiated germanium mold in polyethylene terephthalate (PET), a biocompatible polymer approved by the US Food and Drug Administration for clinical applications, by a novel three-step embossing process. The effects of nano-ripples on Schwann Cells (SCs) are studied in view of their possible use for nerve-repair applications. The data demonstrate that nano-ripples can enhance short-term SC adhesion and proliferation (3-24 h after seeding), drive their actin cytoskeleton spatial organization and sustain long-term cell growth. Notably, SCs are oriented perpendicularly with respect to the nanopattern lines. These results provide information about the possible use of hierarchical nano-rippled elements for nerve-regeneration protocols.

  13. Neuronal Regulation of Schwann Cell Mitochondrial Ca(2+) Signaling during Myelination.

    Science.gov (United States)

    Ino, Daisuke; Sagara, Hiroshi; Suzuki, Junji; Kanemaru, Kazunori; Okubo, Yohei; Iino, Masamitsu

    2015-09-29

    Schwann cells (SCs) myelinate peripheral neurons to promote the rapid conduction of action potentials, and the process of myelination is known to be regulated by signals from axons to SCs. Given that SC mitochondria are one of the potential regulators of myelination, we investigated whether SC mitochondria are regulated by axonal signaling. Here, we show a purinergic mechanism that sends information from neurons to SC mitochondria during myelination. Our results show that electrical stimulation of rat sciatic nerve increases extracellular ATP levels enough to activate purinergic receptors. Indeed, electrical stimulation of sciatic nerves induces Ca(2+) increases in the cytosol and the mitochondrial matrix of surrounding SCs via purinergic receptor activation. Chronic suppression of this pathway during active myelination suppressed the longitudinal and radial development of myelinating SCs and caused hypomyelination. These results demonstrate a neuron-to-SC mitochondria signaling, which is likely to have an important role in proper myelination. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Neuronal Regulation of Schwann Cell Mitochondrial Ca2+ Signaling during Myelination

    Directory of Open Access Journals (Sweden)

    Daisuke Ino

    2015-09-01

    Full Text Available Schwann cells (SCs myelinate peripheral neurons to promote the rapid conduction of action potentials, and the process of myelination is known to be regulated by signals from axons to SCs. Given that SC mitochondria are one of the potential regulators of myelination, we investigated whether SC mitochondria are regulated by axonal signaling. Here, we show a purinergic mechanism that sends information from neurons to SC mitochondria during myelination. Our results show that electrical stimulation of rat sciatic nerve increases extracellular ATP levels enough to activate purinergic receptors. Indeed, electrical stimulation of sciatic nerves induces Ca2+ increases in the cytosol and the mitochondrial matrix of surrounding SCs via purinergic receptor activation. Chronic suppression of this pathway during active myelination suppressed the longitudinal and radial development of myelinating SCs and caused hypomyelination. These results demonstrate a neuron-to-SC mitochondria signaling, which is likely to have an important role in proper myelination.

  15. Glial Cells: The Other Cells of the Nervous System

    Indian Academy of Sciences (India)

    pounded the cell theory with M Schleiden, had diverse interests. ... (Courtesy: Dr. Vanaja Shetty, The Foundation for Medical Research, Mumbai) ... Role of Schwann Cells in Myelination ... arrangement of microvilli extending from the Schwann cell embedded in the gap matrix ... Schwann cells Regulate Nerve Development.

  16. Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer.

    Science.gov (United States)

    Xiao, Lu; Lan, Xiaoying; Shi, Xianping; Zhao, Kai; Wang, Dongrui; Wang, Xuejun; Li, Faqian; Huang, Hongbiao; Liu, Jinbao

    2017-05-18

    Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.

  17. Amorphous areas in the cytoplasm of Dendrobium tepal cells

    Science.gov (United States)

    van Doorn, Wouter G.; Kirasak, Kanjana; Ketsa, Saichol

    2013-01-01

    In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy. PMID:23823702

  18. Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve.

    Science.gov (United States)

    Pereira Lopes, Fátima Rosalina; Frattini, Flávia; Marques, Suelen Adriani; Almeida, Fernanda Martins de; de Moura Campos, Lenira Camargo; Langone, Francesco; Lora, Silvano; Borojevic, Radovan; Martinez, Ana Maria Blanco

    2010-10-01

    Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Nevertheless, the functional outcome after peripheral-nerve regeneration is often poor, especially if the nerve injuries occur far from their targets. Aiming to optimize axon regeneration, we grafted bone-marrow-derived cells (BMDCs) into a collagen-tube nerve guide after transection of the mouse sciatic nerve. The control group received only the culture medium. Motor function was tested at 2, 4, and 6 weeks after surgery, using the sciatic functional index (SFI), and showed that functional recovery was significantly improved in animals that received the cell grafts. After 6 weeks, the mice were anesthetized, perfused transcardially, and the sciatic nerves were dissected and processed for transmission electron microscopy and light microscopy. The proximal and distal segments of the nerves were compared, to address the question of improvement in growth rate; the results revealed a maintenance and increase of nerve regeneration for both myelinated and non-myelinated fibers in distal segments of the experimental group. Also, quantitative analysis of the distal region of the regenerating nerves showed that the numbers of myelinated fibers, Schwann cells (SCs) and g-ratio were significantly increased in the experimental group compared to the control group. The transdifferentiation of BMDCs into Schwann cells was confirmed by double labeling with S100/and Hoechst staining. Our data suggest that BMDCs transplanted into a nerve guide can differentiate into SCs, and improve the growth rate of nerve fibers and motor function in a transected sciatic-nerve model.

  19. Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

    Science.gov (United States)

    Kuang, A.; Musgrave, M. E.

    1996-01-01

    Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

  20. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

  1. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  2. Cytoplasmic Kaiso is associated with poor prognosis in non-small cell lung cancer

    International Nuclear Information System (INIS)

    Dai, Shun-Dong; Wang, Yan; Miao, Yuan; Zhao, Yue; Zhang, Yong; Jiang, Gui-Yang; Zhang, Peng-Xin; Yang, Zhi-Qiang; Wang, En-Hua

    2009-01-01

    Kaiso has been identified as a new member of the POZ-zinc finger family of transcription factors that are implicated in development and cancer. Although controversy still exists, Kaiso is supposed to be involved in human cancer. However, there is limited information regarding the clinical significance of cytoplasmic/nuclear Kaiso in human lung cancer. In this study, immunohistochemical studies were performed on 20 cases of normal lung tissues and 294 cases of non-small cell lung cancer (NSCLC), including 50 cases of paired lymph node metastases and 88 cases with complete follow-up records. Three lung cancer cell lines showing primarily nuclear localization of Kaiso were selected to examine whether roles of Kaiso in cytoplasm and in nucleus are identical. Nuclear Kaiso was down-regulated by shRNA technology or addition a specific Kaiso antibody in these cell lines. The proliferative and invasive abilities were evaluated by MTT and Matrigel invasive assay, transcription of Kaiso's target gene matrilysin was detected by RT-PCR. Kaiso was primarily expressed in the cytoplasm of lung cancer tissues. Overall positive cytoplasmic expression rate was 63.61% (187/294). The positive cytoplasmic expression of Kaiso was higher in advanced TNM stages (III+IV) of NSCLC, compared to lower stages (I+II) (p = 0.019). A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found (p = 0.003). In 50 paired cases, cytoplasmic expression of Kaiso was 78.0% (41/50) in primary sites and 90.0% (45/50) in lymph node metastases (p = 0.001). The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%), compared to those with cytoplasmic Kaiso-negative tumors (64.00%) (p = 0.005). Nuclear Kaiso staining was seen in occasional cases with only a 5.10% (15/294) positive rate and was not associated with any clinicopathological features of NSCLC. Furthermore, after the down-regulation of the nuclear

  3. Hydrodynamic flow in the cytoplasm of plant cells.

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; Lammeren, A.A. van; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

  4. Hydrodynamic flow in the cytoplasm of plant cells

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; van Lammeren, A.A.M.; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

  5. Hydrodynamic flow in the cytoplasm of plant cells

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Houtman, D.; Lammeren, van A.A.M.; Eiser, E.; Emons, A.M.C.

    2008-01-01

    Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study ( Houtman et al., 2007 )

  6. Role of Schwann cells in the regeneration of penile and peripheral nerves

    Directory of Open Access Journals (Sweden)

    Lin Wang

    2015-01-01

    Full Text Available Schwann cells (SCs are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED. Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1 the origin and development of SCs in the peripheral and penile nerve system; (2 Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3 how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4 and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED.

  7. Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

    Science.gov (United States)

    Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

    2017-11-01

    Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

    NARCIS (Netherlands)

    Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm.

  9. A magnetically responsive nanocomposite scaffold combined with Schwann cells promotes sciatic nerve regeneration upon exposure to magnetic field

    Directory of Open Access Journals (Sweden)

    Liu ZY

    2017-10-01

    Full Text Available Zhongyang Liu,1,* Shu Zhu,1,* Liang Liu,2,* Jun Ge,3,4,* Liangliang Huang,1 Zhen Sun,1 Wen Zeng,5 Jinghui Huang,1 Zhuojing Luo1 1Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 2Department of Orthopedics, No 161 Hospital of PLA, Wuhan, Hubei, 3Department of Orthopedics, No 323 Hospital of PLA, Xi’an, Shaanxi, 4Department of Anatomy, Fourth Military Medical University, Xi’an, Shaanxi, 5Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China *These authors contributed equally to this work Abstract: Peripheral nerve repair is still challenging for surgeons. Autologous nerve transplantation is the acknowledged therapy; however, its application is limited by the scarcity of available donor nerves, donor area morbidity, and neuroma formation. Biomaterials for engineering artificial nerves, particularly materials combined with supportive cells, display remarkable promising prospects. Schwann cells (SCs are the absorbing seeding cells in peripheral nerve engineering repair; however, the attenuated biologic activity restricts their application. In this study, a magnetic nanocomposite scaffold fabricated from magnetic nanoparticles and a biodegradable chitosan–glycerophosphate polymer was made. Its structure was evaluated and characterized. The combined effects of magnetic scaffold (MG with an applied magnetic field (MF on the viability of SCs and peripheral nerve injury repair were investigated. The magnetic nanocomposite scaffold showed tunable magnetization and degradation rate. The MGs synergized with the applied MF to enhance the viability of SCs after transplantation. Furthermore, nerve regeneration and functional recovery were promoted by the synergism of SCs-loaded MGs and MF. Based on the current findings, the combined application of MGs and SCs with applied MF is a promising therapy for the engineering of peripheral

  10. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

    OpenAIRE

    Carolina Wählby; Joakim Lindblad; Mikael Vondrus; Ewert Bengtsson; Lennart Björkesten

    2002-01-01

    Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre?processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical ana...

  11. Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

    Science.gov (United States)

    Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

    2015-07-01

    There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

    Science.gov (United States)

    Lee, Ok Ran; Cho, Hyung-Taeg

    2012-12-01

    Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

  13. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    International Nuclear Information System (INIS)

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-01-01

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  14. A new electrospun graphene-silk fibroin composite scaffolds for guiding Schwann cells.

    Science.gov (United States)

    Zhao, Yahong; Gong, Jiahuan; Niu, Changmei; Wei, Ziwei; Shi, Jiaqi; Li, Guohui; Yang, Yumin; Wang, Hongbo

    2017-12-01

    Graphene (Gr) has been made of various forms used for repairing peripheral nerve injury with favorable electroactivity, however, graphene-based scaffolds in peripheral nerve regeneration are still rarely reported due to the difficulty of realizing uniform dispersion of graphene and electroactive materials at nanoscale as well as lacking biocompatibility. In this paper, graphene-silk fibroin (SF) composite nanofiber membranes with different mass ratios were prepared via electrospinning. Microscopic observation revealed that electrospun Gr/SF membranes had a nanofibrous structure. Electrochemical analysis provided electroactivity characterization of the Gr/SF membranes. The physiochemical results showed that the physiochemical properties of electrospun Gr/SF membranes could be changed by varying Gr concentration. Swelling ratio and contact angle measurements confirmed that electrospun Gr/SF membranes possessed large absorption capacity and hydrophilic surface, and the mechanical property was improved with increasing Gr concentration. Additionally, in-vitro cytotoxicity with L929 revealed that all the electrospun Gr/SF membranes are biocompatible. Moreover, the morphology and quantity showed that the membranes supported the survival and growth of the cultured Schwann cells. Collectively, all of the results suggest that the electrospun Gr/SF membranes combine the excellent electrically conductivity and mechanical strength of the graphene with biocompatibility property of silk to mimic the natural neural cell micro-environment for nerve development.

  15. Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

    Science.gov (United States)

    Beilby, Mary J.

    2016-01-01

    The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology. PMID:27504112

  16. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

    Directory of Open Access Journals (Sweden)

    Tomasz J. Wodzicki

    2015-01-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

  17. Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS in Nitella Internodal Cells.

    Directory of Open Access Journals (Sweden)

    Kenji Kikuchi

    Full Text Available Cytoplasmic streaming (CPS is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion.

  18. Cytoplasmic streaming in plant cells emerges naturally by microfilament self-organization.

    Science.gov (United States)

    Woodhouse, Francis G; Goldstein, Raymond E

    2013-08-27

    Many cells exhibit large-scale active circulation of their entire fluid contents, a process termed cytoplasmic streaming. This phenomenon is particularly prevalent in plant cells, often presenting strikingly regimented flow patterns. The driving mechanism in such cells is known: myosin-coated organelles entrain cytoplasm as they process along actin filament bundles fixed at the periphery. Still unknown, however, is the developmental process that constructs the well-ordered actin configurations required for coherent cell-scale flow. Previous experimental works on streaming regeneration in cells of Characean algae, whose longitudinal flow is perhaps the most regimented of all, hint at an autonomous process of microfilament self-organization driving the formation of streaming patterns during morphogenesis. Working from first principles, we propose a robust model of streaming emergence that combines motor dynamics with both microscopic and macroscopic hydrodynamics to explain how several independent processes, each ineffectual on its own, can reinforce to ultimately develop the patterns of streaming observed in the Characeae and other streaming species.

  19. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  20. Grafting of ARPE-19 and Schwann cells to the subretinal space in RCS rats.

    Science.gov (United States)

    Wang, Shaomei; Lu, Bin; Wood, Patrick; Lund, Raymond D

    2005-07-01

    To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

  1. Axon degeneration: make the Schwann cell great again

    Directory of Open Access Journals (Sweden)

    Keit Men Wong

    2017-01-01

    Full Text Available Axonal degeneration is a pivotal feature of many neurodegenerative conditions and substantially accounts for neurological morbidity. A widely used experimental model to study the mechanisms of axonal degeneration is Wallerian degeneration (WD, which occurs after acute axonal injury. In the peripheral nervous system (PNS, WD is characterized by swift dismantling and clearance of injured axons with their myelin sheaths. This is a prerequisite for successful axonal regeneration. In the central nervous system (CNS, WD is much slower, which significantly contributes to failed axonal regeneration. Although it is well-documented that Schwann cells (SCs have a critical role in the regenerative potential of the PNS, to date we have only scarce knowledge as to how SCs 'sense' axonal injury and immediately respond to it. In this regard, it remains unknown as to whether SCs play the role of a passive bystander or an active director during the execution of the highly orchestrated disintegration program of axons. Older reports, together with more recent studies, suggest that SCs mount dynamic injury responses minutes after axonal injury, long before axonal breakdown occurs. The swift SC response to axonal injury could play either a pro-degenerative role, or alternatively a supportive role, to the integrity of distressed axons that have not yet committed to degenerate. Indeed, supporting the latter concept, recent findings in a chronic PNS neurodegeneration model indicate that deactivation of a key molecule promoting SC injury responses exacerbates axonal loss. If this holds true in a broader spectrum of conditions, it may provide the grounds for the development of new glia-centric therapeutic approaches to counteract axonal loss.

  2. α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

    Science.gov (United States)

    Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

    2018-05-02

    α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

  3. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Science.gov (United States)

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  4. Electrically induced brain-derived neurotrophic factor release from Schwann cells.

    Science.gov (United States)

    Luo, Beier; Huang, Jinghui; Lu, Lei; Hu, Xueyu; Luo, Zhuojing; Li, Ming

    2014-07-01

    Regulating the production of brain-derived neurotrophic factor (BDNF) in Schwann cells (SCs) is critical for their application in traumatic nerve injury, neurodegenerative disorders, and demyelination disease in both central and peripheral nervous systems. The present study investigated the possibility of using electrical stimulation (ES) to activate SCs to release BDNF. We found that short-term ES was capable of promoting BDNF production from SCs, and the maximal BDNF release was achieved by ES at 6 V (3 Hz, 30 min). We further examined the involvement of intracellular calcium ions ([Ca2+]i) in the ES-induced BDNF production in SCs by pharmacological studies. We found that the ES-induced BDNF release required calcium influx through T-type voltage-gated calcium channel (VGCC) and calcium mobilization from internal calcium stores, including inositol triphosphate-sensitive stores and caffeine/ryanodine-sensitive stores. In addition, calcium-calmodulin dependent protein kinase IV (CaMK IV), mitogen-activated protein kinase (MAPK), and cAMP response element-binding protein (CREB) were found to play important roles in the ES-induced BDNF release from SCs. In conclusion, ES is capable of activating SCs to secrete BDNF, which requires the involvement of calcium influx through T-type VGCC and calcium mobilization from internal calcium stores. In addition, activation of CaMK IV, MAPK, and CREB were also involved in the ES-induced BDNF release. The findings indicate that ES can improve the neurotrophic ability in SCs and raise the possibility of developing electrically stimulated SCs as a source of cell therapy for nerve injury in both peripheral and central nervous systems. Copyright © 2014 Wiley Periodicals, Inc.

  5. Cellular Subcompartments through Cytoplasmic Streaming.

    Science.gov (United States)

    Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

    2015-08-24

    Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Manipulation of Schwann cell migration across the astrocyte boundary by polysialyltransferase-loaded superparamagnetic nanoparticles under magnetic field

    Directory of Open Access Journals (Sweden)

    Xia B

    2016-12-01

    Full Text Available Bing Xia,* Liangliang Huang,* Lei Zhu, Zhongyang Liu, Teng Ma, Shu Zhu, Jinghui Huang, Zhuojing Luo Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China *These authors contributed equally to this work Abstract: Schwann cell (SC transplantation is an attractive strategy for spinal cord injury (SCI. However, the efficacy of SC transplantation has been limited by the poor migratory ability of SCs in the astrocyte-rich central nervous system (CNS environment and the inability to intermingle with the host astrocyte. In this study, we first magnetofected SCs by polysialyltransferase-functionalized superparamagnetic iron oxide nanoparticles (PST/SPIONs to induce overexpression of polysialylation of neural cell adhesion molecule (PSA-NCAM to enhance SC migration ability, before manipulating the direction of SC migration with the assistance of an applied magnetic field (MF. It was found that magnetofection with PST/SPIONs significantly upregulated the expression of PSA-NCAM in SCs, which significantly enhanced the migration ability of SCs, but without preferential direction in the absence of MF. The number and averaged maximum distance of SCs with PST/SPIONs migrating into the astrocyte domain were significantly enhanced by an applied MF. In a 300 µm row along the astrocyte boundary, the number of SCs with PST/SPIONs migrating into the astrocyte domain under an MF was 2.95 and 6.71 times higher than that in the absence of MF and the intact control SCs, respectively. More interestingly, a confrontation assay demonstrated that SCs with PST/SPIONs were in close contact with astrocytes and no longer formed boundaries in the presence of MF. In conclusion, SCs with PST/SPIONs showed enhanced preferential migration along the axis of a magnetic force, which might be beneficial for the formation of Büngner bands in the CNS. These findings raise the possibilities of enhancing the

  7. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation

    Directory of Open Access Journals (Sweden)

    Keisuke Sato

    2014-01-01

    Full Text Available Epalrestat (EPS, approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH, which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS, the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.

  8. Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents.

    Science.gov (United States)

    Vorob'ev, Vladimir N; Anisimov, Alexander V; Dautova, Nailya R

    2008-07-01

    The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.

  9. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    Science.gov (United States)

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation. © 2016 Federation of European Biochemical Societies.

  10. Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

    Science.gov (United States)

    Girard, Brian J; Regan Anderson, Tarah M; Welch, Siya Lem; Nicely, Julie; Seewaldt, Victoria L; Ostrander, Julie H

    2015-01-01

    Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

  11. Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

    Directory of Open Access Journals (Sweden)

    Ronald Hancock

    Full Text Available Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v or 70 kDa dextran (35% w/v to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox

  12. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

    Science.gov (United States)

    Johannes, E.; Collings, D. A.; Rink, J. C.; Allen, N. S.; Brown, C. S. (Principal Investigator)

    2001-01-01

    In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.

  13. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

    International Nuclear Information System (INIS)

    Liu, Yan; Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui; Wen, Jin-kun

    2013-01-01

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs

  14. Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yan [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); The Third Hospital of Hebei Medical University, Shijazhuang (China); Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); Wen, Jin-kun, E-mail: wjk@hebmu.edu.cn [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China)

    2013-06-28

    Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.

  15. Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors.

    Directory of Open Access Journals (Sweden)

    Ekaterina E Verdiyan

    Full Text Available In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC acetylcholine receptors (AChRs and the axon excitation (different intervals between action potentials (APs. Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the "axon-SC" interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+-influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization.

  16. Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

    DEFF Research Database (Denmark)

    Vogel, L K; Norén, Ove; Sjöström, H

    1995-01-01

    In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance...... of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...

  17. Rac1 controls Schwann cell myelination through cAMP and NF2/merlin

    Science.gov (United States)

    Guo, Li; Moon, Chandra; Niehaus, Karen; Zheng, Yi; Ratner, Nancy

    2013-01-01

    During peripheral nervous system development, Schwann cells (SCs) surrounding single large axons differentiate into myelinating SCs. Previous studies implicate RhoGTPases in SC myelination, but the mechanisms involved in RhoGTPase regulation of SC myelination are unknown. Here, we show that SC myelination is arrested in Rac1 conditional knockout (Rac1-CKO) mice. Rac1 knockout abrogated phosphorylation of the effector p21-activated kinase (PAK) and decreased NF2/merlin phosphorylation. Mutation of NF2/merlin rescued the myelin deficit in Rac1-CKO mice in vivo, and the shortened processes in cultured Rac1-CKO SCs in vitro. Mechanistically, cyclic adenosine monophosphate (cAMP) levels and E-cadherin expression were decreased in the absence of Rac1, and both were restored by mutation of NF2/merlin. Reduced cAMP is a cause of the myelin deficiency in Rac1-CKO mice, as elevation of cAMP by rolipram in Rac1-CKO mice in vivo allowed myelin formation. Thus NF2/merlin and cAMP function downstream of Rac1 signaling in SC myelination, and cAMP levels control Rac1-regulated SC myelination. PMID:23197717

  18. Transfer of vesicles from Schwann cell to axon: a novel mechanism of communication in the peripheral nervous system

    Directory of Open Access Journals (Sweden)

    María Alejandra eLopez-Verrilli

    2012-06-01

    Full Text Available Schwann cells (SCs are the glial component of the peripheral nervous system, with essential roles during development and maintenance of axons, as well as during regenerative processes after nerve injury. SCs increase conduction velocities by myelinating axons, regulate synaptic activity at presynaptic nerve terminals and are a source of trophic factors to neurons. Thus, development and maintenance of peripheral nerves are crucially dependent on local signalling between SCs and axons. In addition to the classic mechanisms of intercellular signalling, the possibility of communication through secreted vesicles has been poorly explored to date. Interesting recent findings suggest the occurrence of lateral transfer mediated by vesicles from glial cells to axons that could have important roles in axonal growth and axonal regeneration. Here, we review the role of vesicular transfer from SCs to axons and propose the benefits of this means in supporting neuronal and axonal maintenance and regeneration after nerve damage.

  19. Skin derived precursor Schwann cell-generated acellular matrix modified chitosan/silk scaffolds for bridging rat sciatic nerve gap.

    Science.gov (United States)

    Zhu, Changlai; Huang, Jing; Xue, Chengbin; Wang, Yaxian; Wang, Shengran; Bao, Shuangxi; Chen, Ruyue; Li, Yuan; Gu, Yun

    2017-12-27

    Extracellular/acellular matrix has been attracted much research interests for its unique biological characteristics, and ACM modified neural scaffolds shows the remarkable role of promoting peripheral nerve regeneration. In this study, skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) were used as parent cells to generate acellular(ACM) for constructing a ACM-modified neural scaffold. SKP-SCs were co-cultured with chitosan nerve guidance conduits (NGC) and silk fibroin filamentous fillers, followed by decellularization to stimulate ACM deposition. This NGC-based, SKP-SC-derived ACM-modified neural scaffold was used for bridging a 10 mm long rat sciatic nerve gap. Histological and functional evaluation after grafting demonstrated that regenerative outcomes achieved by this engineered neural scaffold were better than those achieved by a plain chitosan-silk fibroin scaffold, and suggested the benefits of SKP-SC-derived ACM for peripheral nerve repair. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  20. Switchable cell trapping using superparamagnetic beads

    Energy Technology Data Exchange (ETDEWEB)

    Bryan, M. T.; Smith, K. H.; Real, M. E.; Bashir, M. A.; Fry, P. W.; Fischer, P.; Im, M.-Y.; Schrefl, T.; Allwood, D. A.; Haycock, J. W.

    2010-04-30

    Ni{sub 81}Fe{sub 19} microwires are investigated as the basis of a switchable template for positioning magnetically-labeled neural Schwann cells. Magnetic transmission X-ray microscopy and micromagnetic modeling show that magnetic domain walls can be created or removed in zigzagged structures by an applied magnetic field. Schwann cells containing superparamagnetic beads are trapped by the field emanating from the domain walls. The design allows Schwann cells to be organized on a surface to form a connected network and then released from the surface if required. As aligned Schwann cells can guide nerve regeneration, this technique is of value for developing glial-neuronal co-culture models in the future treatment of peripheral nerve injuries.

  1. The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    International Nuclear Information System (INIS)

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2005-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

  2. La célula de Schwann

    Directory of Open Access Journals (Sweden)

    Adriana del Pilar López Lombana

    1993-12-01

    Full Text Available La célula de Schwann que constituye la glía del SNP, además de ser el soporte estructural para los axones en dicho sistema, tiene la función de producir la mielina, una organela de gran importancia en los procesos de neuroconducción. De la integridad de esta célula dependen el desarrollo estructural y metabólico del axón, así mismo se ha reconocido desde hace varios anos el papel primordial que juega ella, en los procesos de regeneración del SPN posterior a una injuria, en cuyo caso reinician la proliferación para producir una guía de regeneración del nervio periférico. En esta revisión se contemplarán algunos de los puntos relacionados con su origen, desarrollo, estructura, relación con el axon y el tipo de patologías que pueden alterarla; igualmente se resalta la utilidad de los cultivos de celulas de Schwann para el estudio de los procesos de mielinización, desmielinización, regeneración post-traumatica y respuesta a agentes infecciosos.

  3. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Takahashi

    Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

  4. Cytoplasmic chromatin triggers inflammation in senescence and cancer.

    Science.gov (United States)

    Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

    2017-10-19

    Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

  5. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  6. Regulated viral BDNF delivery in combination with Schwann cells promotes axonal regeneration through capillary alginate hydrogels after spinal cord injury.

    Science.gov (United States)

    Liu, Shengwen; Sandner, Beatrice; Schackel, Thomas; Nicholson, LaShae; Chtarto, Abdelwahed; Tenenbaum, Liliane; Puttagunta, Radhika; Müller, Rainer; Weidner, Norbert; Blesch, Armin

    2017-09-15

    Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord. Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within

  7. Cytoplasmic influence of nucleolar development

    International Nuclear Information System (INIS)

    Ghosh, Sibdas

    1974-01-01

    The role of cytoplasmic factors on the development of nucleolus in nucleus has been investigated in Ehrlich mouse ascites tumour cells using tritiated thymidine/uridine for autoradiography. It is inferred from the observations that the cytoplasmic factors has some but not absolute control over the development of nucleolus. (M.G.B.)

  8. Cytoplasmic inheritance of parent-offspring cell structure in the clonal diatom Cyclotella meneghiniana.

    Science.gov (United States)

    Shirokawa, Yuka; Shimada, Masakazu

    2016-11-16

    In cytoplasmic inheritance, structural states of a parent cell could be transmitted to offspring cells via two mechanisms. The first is referred to as the hangover of parent structure, where the structure itself remains and faithfully transmits within offspring cells; the second is structural inheritance, wherein the parent structure functions as a template for development of new offspring structure. We estimated to what extent the parent structure affects the development of offspring structure by structural inheritance, using a clone of the diatom Cyclotella meneghiniana The cell has two siliceous valves (a cell wall part at both cell poles): one is inherited from the parent and the other is newly formed. We estimated cytoplasmic heritability by comparing valve traits (central fultoportulae (CTFP), striae, central area, and cell diameter) of parent and new offspring valves, using single-cell isolation and valve labelling. Parent-offspring valve trait regressions showed that all traits, except CTFP, were significantly correlated. We formulated a quantitative genetic model considering the diatom inheritance system and revealed short-term rapid evolution compared with other inheritance systems. Diatom structural inheritance will have evolved to enable clonal populations to rapidly acquire and maintain suitable structures for temporal changes in environments and life-cycle stages. © 2016 The Author(s).

  9. Mechanisms for cytoplasmic organization: an overview.

    Science.gov (United States)

    Pagliaro, L

    2000-01-01

    One of the basic characteristics of life is the intrinsic organization of cytoplasm, yet we know surprisingly little about the manner in which cytoplasmic macromolecules are arranged. It is clear that cytoplasm is not the homogeneous "soup" it was once envisioned to be, but a comprehensive model for cytoplasmic organization is not available in modern cell biology. The premise of this volume is that phase separation in cytoplasm may play a role in organization at the subcellular level. Other mechanisms for non-membrane-bounded intracellular organization have previously been proposed. Some of these will be reviewed in this chapter. Multiple mechanisms, involving phase separation, specific intracellular targeting, formation of macromolecular complexes, and channeling, all could well contribute to cytoplasmic organization. Temporal and spatial organization, as well as composition, are likely to be important in defining the characteristics of cytoplasm.

  10. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  11. Electroactive biodegradable polyurethane significantly enhanced Schwann cells myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering.

    Science.gov (United States)

    Wu, Yaobin; Wang, Ling; Guo, Baolin; Shao, Yongpin; Ma, Peter X

    2016-05-01

    Myelination of Schwann cells (SCs) is critical for the success of peripheral nerve regeneration, and biomaterials that can promote SCs' neurotrophin secretion as scaffolds are beneficial for nerve repair. Here we present a biomaterials-approach, specifically, a highly tunable conductive biodegradable flexible polyurethane by polycondensation of poly(glycerol sebacate) and aniline pentamer, to significantly enhance SCs' myelin gene expression and neurotrophin secretion for peripheral nerve tissue engineering. SCs are cultured on these conductive polymer films, and the biocompatibility of these films and their ability to enhance myelin gene expressions and sustained neurotrophin secretion are successfully demonstrated. The mechanism of SCs' neurotrophin secretion on conductive films is demonstrated by investigating the relationship between intracellular Ca(2+) level and SCs' myelination. Furthermore, the neurite growth and elongation of PC12 cells are induced by adding the neurotrophin medium suspension produced from SCs-laden conductive films. These data suggest that these conductive degradable polyurethanes that enhance SCs' myelin gene expressions and sustained neurotrophin secretion perform great potential for nerve regeneration applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    Science.gov (United States)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  13. Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

    Science.gov (United States)

    Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

    2014-10-15

    Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. © 2014 Lechuga, Baranwal, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

    Science.gov (United States)

    Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

    2011-01-01

    Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

  15. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  16. Cytoplasmic pH and the regulation of the dictyostelium cell cycle

    NARCIS (Netherlands)

    Aerts, R.J.; Durston, A.J.; Moolenaar, W.H.

    1985-01-01

    Cytoplasmic pH (pHl) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH “null point” method. There is a cycle of pHl that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S

  17. Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells.

    Science.gov (United States)

    Huang, Jinghui; Ye, Zhengxu; Hu, Xueyu; Lu, Lei; Luo, Zhuojing

    2010-04-01

    Production of nerve growth factor (NGF) from Schwann cells (SCs) progressively declines in the distal stump, if axonal regeneration is staggered across the suture site after peripheral nerve injuries. This may be an important factor limiting the outcome of nerve injury repair. Thus far, extensive efforts are devoted to modulating NGF production in cultured SCs, but little has been achieved. In the present in vitro study, electrical stimulation (ES) was attempted to stimulate cultured SCs to release NGF. Our data showed that ES was capable of enhancing NGF release from cultured SCs. An electrical field (1 Hz, 5 V/cm) caused a 4.1-fold increase in NGF release from cultured SCs. The ES-induced NGF release is calcium dependent. Depletion of extracellular or/and intracellular calcium partially/ completely abolished the ES-induced NGF release. Further pharmacological interventions showed that ES induces calcium influx through T-type voltage-gated calcium channels and mobilizes calcium from 1, 4, 5-trisphosphate-sensitive stores and caffeine/ryanodine-sensitive stores, both of which contributed to the enhanced NGF release induced by ES. In addition, a calcium-triggered exocytosis mechanism was involved in the ES-induced NGF release from cultured SCs. These findings show the feasibility of using ES in stimulating SCs to release NGF, which holds great potential in promoting nerve regeneration by enhancing survival and outgrowth of damaged nerves, and is of great significance in nerve injury repair and neuronal tissue engineering.

  18. Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers

    NARCIS (Netherlands)

    Ketelaar, T.; Honing, van der H.S.; Emons, A.M.C.

    2010-01-01

    In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be

  19. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Directory of Open Access Journals (Sweden)

    Lauren Sweet

    Full Text Available Numerous studies have demonstrated that Schwann cells (SCs play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP scaffolds arranged in 3D printed-lattice (P-β-TCP and randomly-porous, template-casted (N-β-TCP structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf, neutrophin-3 (nt-3, platelet-derived growth factor (pdgf-bb, and vascular endothelial growth factor (vegf-a were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  20. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    International Nuclear Information System (INIS)

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-01-01

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

  1. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  2. A history of plant biotechnology: from the Cell Theory of Schleiden and Schwann to biotech crops.

    Science.gov (United States)

    Vasil, Indra K

    2008-09-01

    Plant biotechnology is founded on the principles of cellular totipotency and genetic transformation, which can be traced back to the Cell Theory of Matthias Jakob Schleiden and Theodor Schwann, and the discovery of genetic transformation in bacteria by Frederick Griffith, respectively. On the 25th anniversary of the genetic transformation of plants, this review provides a historical account of the evolution of the theoretical concepts and experimental strategies that led to the production and commercialization of biotech (transformed or transgenic) plants expressing many useful genes, and emphasizes the beneficial effects of plant biotechnology on food security, human health, the environment, and conservation of biodiversity. In so doing, it celebrates and pays tribute to the contributions of scores of scientists who laid the foundation of modern plant biotechnology by their bold and unconventional thinking and experimentation. It highlights also the many important lessons to be learnt from the fascinating history of plant biotechnology, the significance of history in science teaching and research, and warns against the danger of the growing trends of ignoring history and historical illiteracy.

  3. Combined effects of rat Schwann cells and 17β-estradiol in a spinal cord injury model.

    Science.gov (United States)

    Namjoo, Zeinab; Moradi, Fateme; Aryanpour, Roya; Piryaei, Abbas; Joghataei, Mohammad Taghi; Abbasi, Yusef; Hosseini, Amir; Hassanzadeh, Sajad; Taklimie, Fatemeh Ranjbar; Beyer, Cordian; Zendedel, Adib

    2018-04-15

    Spinal cord injury (SCI) is a devastating traumatic event which burdens the affected individuals and the health system. Schwann cell (SC) transplantation is a promising repair strategy after SCI. However, a large number of SCs do not survive following transplantation. Previous studies demonstrated that 17β-estradiol (E2) protects different cell types and reduces tissue damage in SCI experimental animal model. In the current study, we evaluated the protective potential of E2 on SCs in vitro and investigated whether the combination of hormonal and SC therapeutic strategy has a better effect on the outcome after SCI. Primary SC cultures were incubated with E2 for 72 h. In a subsequent experiment, thoracic contusion SCI was induced in male rats followed by sustained administration of E2 or vehicle. Eight days after SCI, DiI-labeled SCs were transplanted into the injury epicenter in vehicle and E2-treated animals. The combinatory regimen decreased neurological and behavioral deficits and protected neurons and oligodendrocytes in comparison to vehicle rats. Moreover, E2 and SC significantly decreased the number of Iba-1+ (microglia) and GFAP + cells (astrocyte) in the SCI group. In addition, we found a significant reduction of mitochondrial fission-markers (Fis1) and an increase of fusion-markers (Mfn1 and Mfn2) in the injured spinal cord after E2 and SC treatment. These data demonstrated that E2 protects SCs against hypoxia-induced SCI and improves the survival of transplanted SCs.

  4. Implications of Schwann Cells Biomechanics and Mechanosensitivity for Peripheral Nervous System Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Gonzalo Rosso

    2017-10-01

    Full Text Available The presence of bones around the central nervous system (CNS provides it with highly effective physiologically crucial mechanical protection. The peripheral nervous system (PNS, in contrast, lacks this barrier. Consequently, the long held belief is that the PNS is mechanically vulnerable. On the other hand, the PNS is exposed to a variety of physiological mechanical stresses during regular daily activities. This fact prompts us to question the dogma of PNS mechanical vulnerability. As a matter of fact, impaired mechanics of PNS nerves is associated with neuropathies with the liability to mechanical stresses paralleled by significant impairment of PNS physiological functions. Our recent biomechanical integrity investigations on nerve fibers from wild-type and neuropathic mice lend strong support in favor of natural mechanical protection of the PNS and demonstrate a key role of Schwann cells (SCs therein. Moreover, recent works point out that SCs can sense mechanical properties of their microenvironment and the evidence is growing that SCs mechanosensitivity is important for PNS development and myelination. Hence, SCs exhibit mechanical strength necessary for PNS mechanoprotection as well as mechanosensitivity necessary for PNS development and myelination. This mini review reflects on the intriguing dual ability of SCs and implications for PNS physiology and pathophysiology.

  5. Combination Therapy with c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells.

    Science.gov (United States)

    Fuse, Marisa A; Plati, Stephani Klingeman; Burns, Sarah S; Dinh, Christine T; Bracho, Olena; Yan, Denise; Mittal, Rahul; Shen, Rulong; Soulakova, Julia N; Copik, Alicja J; Liu, Xue Zhong; Telischi, Fred F; Chang, Long-Sheng; Franco, Maria Clara; Fernandez-Valle, Cristina

    2017-11-01

    Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G 1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387-98. ©2017 AACR . ©2017 American Association for Cancer Research.

  6. Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

    Science.gov (United States)

    Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

    2015-06-01

    It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

  7. Formation and function of synapses with respect to Schwann cells at the end of motor nerve terminal branches on mature amphibian (Bufo marinus) muscle.

    Science.gov (United States)

    Macleod, G T; Dickens, P A; Bennett, M R

    2001-04-01

    A study has been made of the formation and regression of synapses with respect to Schwann cells at the ends of motor nerve terminal branches in mature toad (Bufo marinus) muscle. Synapse formation and regression, as inferred from the appearance and loss of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43)-stained vesicle clusters, occurred at the ends of terminal branches over a 16 hr period. Multiple microelectrodes placed in an array about FM1-43 blobs at the ends of terminal branches detected the electrical signs of neurotransmitter being released onto receptors. Injection of a calcium indicator (Oregon Green 488 BAPTA-1) into the motor nerve with subsequent imaging of the calcium transients, in response to stimulation, often showed a reduced calcium influx in the ends of terminal branches. Injection of a fluorescent dye into motor nerves revealed the full extent of their terminal branches and growing processes. Injection of the terminal Schwann cells (TSCs) often revealed pseudopodial TSC processes up to 10-microm-long. Imaging of these TSC processes over minutes or hours showed that they were highly labile and capable of extending several micrometers in a few minutes. Injection of motor nerve terminals with a different dye to that injected into their TSCs revealed that terminal processes sometimes followed the TSC processes over a few hours. It is suggested that the ends of motor nerve terminals in vivo are in a constant state of remodeling through the formation and regression of processes, that TSC processes guide the remodeling, and that it can occur over a relatively short period of time.

  8. Cation binding at the node of Ranvier: I. Localization of binding sites during development.

    Science.gov (United States)

    Zagoren, J C; Raine, C S; Suzuki, K

    1982-06-17

    Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

  9. Glial Cells: The Other Cells of the Nervous System

    Indian Academy of Sciences (India)

    Theodor Schwann, the German physiologist who first pro- pounded the cell theory with M Schleiden, had diverse interests. He was not only the first to isolate the enzyme pepsin, but also investigated muscle contraction and nerve structure. In the mid nineteenth century Schwann discovered that a sheath made up of myelin ...

  10. A fine structural localization of the non-specific cholinesterase activity in glomerular nerve formations (endings).

    Science.gov (United States)

    Dubový, P

    1990-01-01

    Snout glabrous skin (rhinarium) of the cat is innervated not only by typical simple lamellar corpuscles but also glomerular formations. In contrast to simple lamellar corpuscles, glomerular nerve formations are located away the dermal papillae. In cross sections, glomerular nerve formation consists of several axonal profiles enveloped by 1-2 cytoplasmic lamellae of Schwann cells. The space among them is filled by collagenous microfibrils and the basal lamina-like material. Capsule was composed from fibroblast-like cells without definite basal lamina. An electron-dense reaction product due to non-specific cholinesterase activity was associated with Schwann cells and their processes surrounding unmyelinated terminal portion of the sensory axons. Abundant reaction product was bound to the collagenous microfibrils and was deposited in extracellular matrix between Schwann cell processes. These results are further evidence for the presence of the non-specific cholinesterase molecules as integral component of the extracellular matrix in sensory corpuscles. On the basis of histochemical study two possible explanation are considered for functional involving of this enzyme in sensory nerve formations.

  11. Single-walled carbon nanotubes alter Schwann cell behavior differentially within 2D and 3D environments.

    Science.gov (United States)

    Behan, Brenda L; DeWitt, Daniel G; Bogdanowicz, Danielle R; Koppes, Abigail N; Bale, Shyam S; Thompson, Deanna M

    2011-01-01

    Both spinal cord injury (SCI) and large-gap peripheral nerve defects can be debilitating affecting a patient's long-term quality of life and presently, there is no suitable treatment for functional regeneration of these injured tissues. A number of works have suggested the benefits of electrical stimulation to promote both glial migration and neuronal extension. In this work, an electrically conductive hydrogel containing single-walled carbon nanotubes (SWCNT) for neural engineering applications is presented and the Schwann cell (SC) response to SWCNT is examined in both 2D and 3D microenvironments. Results from clonogenic and alamarBlue® assays in 2D indicate that SWCNT (10-50 μg mL(-1)) inhibit SC proliferation but do not affect cell viability. Following SWCNT exposure in 2D, changes in SC morphology can be observed with the nanomaterial attached to the cell membrane at concentrations as low as 10 μg mL(-1). In contrast to the results gathered in 2D, SC embedded within the 3D hydrogel loaded with 10-50 μg mL(-1) of SWCNT exhibited little or no measurable change in cell proliferation, viability, or morphology as assessed using a digestion assay, alamarBlue, and confocal microscopy. Collectively, this highlights that an electrically-conductive SWCNT collagen I-Matrigel™ biomaterial may be suitable for neural tissue engineering and is able to sustain populations of SC. Findings suggest that 2D nanoparticle toxicity assays may not be accurate predictors of the 3D response, further motivating the examination of these materials in a more physiologically relevant environment. Copyright © 2010 Wiley Periodicals, Inc.

  12. Macrophage-derived microvesicles promote proliferation and migration of Schwann cell on peripheral nerve repair

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, Chuan, E-mail: zhchuansy@163.com; Ma, Cheng-bin; Yuan, Hong-mou; Cao, Bao-yuan; Zhu, Jia-jun

    2015-12-04

    Background: Macrophages have been implicated in peripheral nerve regeneration. However, whether macrophages-derived microvesicles (MVs) are involved in this process remains unknown. In the present study, the effects of macrophages-derived MVs on proliferation and migration of Schwann cells (SCs) were evaluated in both in vitro and in vivo. Methods: Human monocytic leukaemia cell line (THP-1) was successfully driven to M1 and M2 phenotypes by delivery of either IFN-γ or IL-4, respectively. SCs incubated with M1 or M2 macrophages-derived MVs, the cell migration and proliferation were assessed, and expression levels of nerve growth factor (NGF) and Laminin were measured. A rat model of sciatic nerve was established and the effects of macrophages-derived MVs on nerve regeneration were investigated. Results: M2-derived MVs elevated migration, proliferation, NFG and Laminin protein levels of SCs compared with M1-or M0-derived MVs. The relative expression levels of miR-223 were also increased in M2 macrophages and M2-derived MVs. Transfected M2 macrophages with miR-223 inhibitor then co-incubated with SCs, an inhibition of cell migration and proliferation and a down-regulated levels of NFG and Laminin protein expression were observed. In vivo, M2-derived MVs significantly increased the infiltration and axon number of SCs. Conclusion: M2-derived MVs promoted proliferation and migration of SCs in vitro and in vivo, which provided a therapeutic strategy for nerve regeneration. - Highlights: • M2 macrophages-derived MVs elevated migration and proliferation of SCs. • M2 macrophages-derived MVs up-regulated NFG and Laminin expression of SCs. • MiR-223 expression was increased in M2 macrophages-derived MVs. • MiR-223 inhibitor reduced migration and proliferation of SCs co-incubated with MVs. • MiR-223 inhibitor down-regulated NFG and Laminin levels of SCs co-incubated with MVs.

  13. Mechanosensitivity of Embryonic Neurites Promotes Their Directional Extension and Schwann Cells Progenitors Migration

    Directory of Open Access Journals (Sweden)

    Gonzalo Rosso

    2017-11-01

    Full Text Available Background/Aims: Migration of Schwann cells (SCs progenitors and neurite outgrowth from embryonic dorsal root ganglions (DRGs are two central events during the development of the peripheral nervous system (PNS. How these two enthralling events preceding myelination are promoted is of great relevance from basic research and clinical aspects alike. Recent evidence demonstrates that biophysical cues (extracellular matrix stiffness and biochemical signaling act in concert to regulate PNS myelination. Microenvironment stiffness of SCs progenitors and embryonic neurites dynamically changes during development. Methods: DRG explants were isolated from day 12.5 to 13.5 mice embryos and plated on laminin-coated substrates with varied stiffness values. After 4 days in culture and immunostaining with specific markers, neurite outgrowth pattern, SCs progenitors migration, and growth cone shape and advance were analyzed with confocal fluorescence microscopy. Results: We found out that growing substrate stiffness promotes directional neurite outgrowth, SCs progenitors migration, growth cone advance and presumably axons fasciculation. Conclusions: DRG explants are in vitro models for the research of PNS development, myelination and regeneration. Consequently, we conclude the following: Our observations point out the importance of mechanosensitivity for the PNS. At the same time, they prompt the investigation of the important yet unclear links between PNS biomechanics and inherited neuropathies with myelination disorders such as Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies. Finally, they encourage the consideration of mechanosensitivity in bioengineering of scaffolds to aid nerve regeneration after injury.

  14. The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

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    Deepshi Thakral

    Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

  15. Making it big : how characean algae use cytoplasmic streaming to enhance transport in giant cells

    NARCIS (Netherlands)

    Meent, Jan Willem van de

    2010-01-01

    Organisms show a remarkable variation in sizes, yet cell sizes are surprisingly similar across species, typically ranging from 10 μm to 100 μm. A striking exception are the giant cells of the algal weed Chara, which can exceed 10 cm in length and 1 mm in diameter. A circulation known as cytoplasmic

  16. Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

    Science.gov (United States)

    Chen, Tai-Yen; Jung, Won; Santiago, Ace George; Yang, Feng; Krzemiński, Łukasz; Chen, Peng

    2015-11-12

    Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

  17. Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

    International Nuclear Information System (INIS)

    Heude, M.; Chanet, R.

    1975-01-01

    The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid-held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin and chloramphenicol. It was shown that mitochondrial proteins are involved in the recovery and survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the e + genotype in UV-irradiated dark liquid-held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid-holding process for the e - induction, as shown by inhibiting mitochondrial protein synthesis of both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid-holding of the UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage in particular is not correlated with the repressed or derepressed state of the mitochondria

  18. 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. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Cytoplasmic Estrogen Receptor in breast cancer

    Science.gov (United States)

    Welsh, Allison W.; Lannin, Donald R.; Young, Gregory S.; Sherman, Mark E.; Figueroa, Jonine D.; Henry, N. Lynn; Ryden, Lisa; Kim, Chungyeul; Love, Richard R.; Schiff, Rachel; Rimm, David L.

    2011-01-01

    Purpose In addition to genomic signaling, it is accepted that ERα has non-nuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of non-nuclear ER in clinical specimens. Experimental Design A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, 1D5) were validated by western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. Results Four of the antibodies specifically recognized ER by western and QIF, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with pearson’s correlations (r2 values)ranging from 0.87-0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other 4 antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. Conclusions Our data shows ERα present in the cytoplasm in a number of cases using multiple antibodies, while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value. PMID:21980134

  20. Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

    International Nuclear Information System (INIS)

    Symillides, Y.

    1985-09-01

    An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14 C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

  1. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  2. Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

    Science.gov (United States)

    Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

    2013-01-01

    The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

  3. Tang-Luo-Ning, a Traditional Chinese Medicine, Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis of Schwann Cells under High Glucose Environment

    Directory of Open Access Journals (Sweden)

    Weijie Yao

    2017-01-01

    Full Text Available Tang-Luo-Ning (TLN has a definite effect in the clinical treatment of diabetic peripheral neuropathy (DPN. Schwann cells (SCs apoptosis induced by endoplasmic reticulum stress (ER stress is one of the main pathogeneses of DPN. This study investigates whether TLN can inhibit SCs apoptosis by inhibiting ER stress-induced apoptosis. Our previous researches have demonstrated that TLN could increase the expression of ER stress marker protein GRP78 and inhibited the expression of apoptosis marker protein CHOP in ER stress. In this study, the results showed that TLN attenuated apoptosis by decreasing Ca2+ level in SCs and maintaining ER morphology. TLN could decrease downstream proteins of CHOP including GADD34 and Ero1α, while it increased P-eIF2α and decreased the upstream proteins of CHOP including P-IRE1α/IRE1α and XBP-1, thereby reducing ER stress-induced apoptosis.

  4. Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell Myelination, Are Responsible for Arthrogryposis Multiplex Congenita.

    Science.gov (United States)

    Xue, Shifeng; Maluenda, Jérôme; Marguet, Florent; Shboul, Mohammad; Quevarec, Loïc; Bonnard, Carine; Ng, Alvin Yu Jin; Tohari, Sumanty; Tan, Thong Teck; Kong, Mung Kei; Monaghan, Kristin G; Cho, Megan T; Siskind, Carly E; Sampson, Jacinda B; Rocha, Carolina Tesi; Alkazaleh, Fawaz; Gonzales, Marie; Rigonnot, Luc; Whalen, Sandra; Gut, Marta; Gut, Ivo; Bucourt, Martine; Venkatesh, Byrappa; Laquerrière, Annie; Reversade, Bruno; Melki, Judith

    2017-04-06

    Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  5. Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

    OpenAIRE

    Puddington, L; Woodgett, C; Rose, J K

    1987-01-01

    The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

  6. La célula de schwann

    OpenAIRE

    Perdomo, Sandra; Spinel, Clara

    2011-01-01

    Las neuronas son las células del sistema nervioso y están recubiertas y protegidas por células gliales. En el sistema nerviosos periférico las células de Schwann (CS) son la glía de los nervios. Las prolongaciones o neuritas (axón y dendrita) de los cuerpos de las neuronas son recubiertas por las CS y constituyen las fibras nerviosas. La relación íntima entre la CS y la neurita se determina durante el desarrollo embrionario. La CS es esencial en la migración correcta de las neuritas hacia su ...

  7. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  8. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    Science.gov (United States)

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  9. Glial cell biology in the Great Lakes region.

    Science.gov (United States)

    Feinstein, Douglas L; Skoff, Robert P

    2016-03-31

    We report on the tenth bi-annual Great Lakes Glial meeting, held in Traverse City, Michigan, USA, September 27-29 2015. The GLG meeting is a small conference that focuses on current research in glial cell biology. The array of functions that glial cells (astrocytes, microglia, oligodendrocytes, Schwann cells) play in health and disease is constantly increasing. Despite this diversity, GLG meetings bring together scientists with common interests, leading to a better understanding of these cells. This year's meeting included two keynote speakers who presented talks on the regulation of CNS myelination and the consequences of stress on Schwann cell biology. Twenty-two other talks were presented along with two poster sessions. Sessions covered recent findings in the areas of microglial and astrocyte activation; age-dependent changes to glial cells, Schwann cell development and pathology, and the role of stem cells in glioma and neural regeneration.

  10. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    International Nuclear Information System (INIS)

    Kutty, R. Krishnan; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-01-01

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 27 KKRKAP 276 ) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting

  11. Curcumin accelerates the repair of sciatic nerve injury in rats through reducing Schwann cells apoptosis and promoting myelinization.

    Science.gov (United States)

    Zhao, Zhiwei; Li, Xiaoling; Li, Qing

    2017-08-01

    Schwann cells (SCs) play an indispensable role in the repair and regeneration of injured peripheral nerve. Curcumin can reduce SCs apoptosis, and promote the regeneration and functional recovery of injured peripheral nerves. However, the corresponding mechanisms are not clear. The article was aimed to explore the effect and corresponding mechanisms of curcumin on the repair of sciatic nerve injury in rats. After surgery induced sciatic nerve injury, the model rats were divided into three groups and treated with curcumin, curcumin+PD98059 and curcumin+IGF-1 respectively for 4days. The phosphorylation of Erk1/2 and Akt, and the expression of LC3-II, Beclin 1 and p62 were measured using western blotting. After treatment for 60days, myelination of the injured sciatic nerve was evaluated by MBP immunohistochemical staining and the expression of PMP22, Fibrin and S100 were determined using qRT-PCR and western blotting. In vitro, RSC96 cells were starved for 12h to induce autophagy, and received DMSO, curcumin, PD98059+curcumin, IGF-1+curcumin and BFA1 respectively. The phosphorylation of Erk1/2、Akt and the expression of LC3-II, Beclin 1, p62, PMP22, Fibrin and S100 were measured using western blotting, and the cell apoptosis was detected by flow cytometry. Curcumin could promote injury-induced cell autophagy, remyelination and axon regeneration in sciatic nerve of rats. In vitro, curcumin could accelerate cell autophagy through regulating autophagy related Erk1/2 and Akt pathway, prevent cell apoptosis and promote expression of PMP22 and S100, and reduced deposition of Fibrin in cultured RSC96 SCs. Curcumin could accelerate injured sciatic nerve repair in rats through reducing SCs apoptosis and promoting myelinization. Copyright © 2017. Published by Elsevier Masson SAS.

  12. Effect of external pH on the cytoplasmic and vacuolar pHs in Mung bean root-tip cells

    International Nuclear Information System (INIS)

    Torimitsu, Keiichi; Yazaki, Yoshiaki; Nagasuka, Kinuyo; Ohta, Eiji; Sakata, Makoto

    1984-01-01

    The effect of the external pH on the intracellular pH in mung bean (Vigna mungo (L.) Hepper) root-tip cells was investigated with the 31 P nuclear magnetic resonance (NMR) method. The 31 P NMR spectra showed three peaks caused by cytoplasmic G-6-P, cytoplasmic Psub(i) and vacuolar Psub(i). The cytoplasmic and vacuolar pHs could be determined by comparing the Psub(i) chemical shifts with the titration curve. When the external pH was changed over a range from pH 3 to 10, the cytoplasmic pH showed smaller changes than the vacuolar pH, suggesting that the former is regulated more strictly than the latter. The H + -ATPase inhibitor, DCCD, caused the breakdown of the mechanism that regulates the intracellular pH. H + -ATPase appears to have an important part in the regulation of the intracellular pH. (author)

  13. The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca2+-dependent pathway(s)

    International Nuclear Information System (INIS)

    Manzati, Elisa; Aguiari, Gianluca; Banzi, Manuela; Manzati, Michele; Selvatici, Rita; Falzarano, Sofia; Maestri, Iva; Pinton, Paolo; Rizzuto, Rosario; Senno, Laura del

    2005-01-01

    Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca 2+ levels and activation of PKCα, thereby upregulating D1 and D3 cyclin, downregulating p21 waf1 and p27 kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca 2+ increase and PKCα activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKCα or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca 2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation

  14. How crowded is the prokaryotic cytoplasm?

    NARCIS (Netherlands)

    Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

    2013-01-01

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

  15. Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

    Science.gov (United States)

    Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

    2005-10-20

    The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

  16. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  17. The molecular mechanism and physiological role of cytoplasmic streaming.

    Science.gov (United States)

    Tominaga, Motoki; Ito, Kohji

    2015-10-01

    Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

    Science.gov (United States)

    Haukland, H H; Ulvatne, H; Sandvik, K; Vorland, L H

    2001-11-23

    The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

  19. Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes

    Science.gov (United States)

    Christie, Joshua R.; Beekman, Madeleine

    2017-01-01

    Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution. PMID:28025277

  20. Navigating neurites utilize cellular topography of Schwann cell somas and processes for optimal guidance

    Science.gov (United States)

    Lopez-Fagundo, Cristina; Mitchel, Jennifer A.; Ramchal, Talisha D.; Dingle, Yu-Ting L.; Hoffman-Kim, Diane

    2013-01-01

    The path created by aligned Schwann cells (SCs) after nerve injury underlies peripheral nerve regeneration. We developed geometric bioinspired substrates to extract key information needed for axon guidance by deconstructing the topographical cues presented by SCs. We have previously reported materials that directly replicate SC topography with micro- and nanoscale resolution, but a detailed explanation of the means of directed axon extension on SC topography has not yet been described. Here, using neurite tracing and time-lapse microscopy, we analyzed the SC features that influence axon guidance. Novel poly(dimethylsiloxane) materials, fabricated via photolithography, incorporated bioinspired topographical components with the shapes and sizes of aligned SCs, namely somas and processes, where the length of the processes were varied but the soma geometry and dimensions were kept constant. Rat dorsal root ganglia neurites aligned to all materials presenting bioinspired topography after a 5 days in culture and to bioinspired materials presenting soma and process features after only 17 hours in culture. Key findings of this study were: Neurite response to underlying bioinspired topographical features was time dependent, where at 5 days, neurites aligned most strongly to materials presenting combinations of soma and process features, with higher than average density of either process or soma features; but at 17 hours they aligned more strongly to materials presenting average densities of soma and process features and to materials presenting process features only. These studies elucidate the influence of SC topography on axon guidance in a time-dependent setting and have implications for the optimization of nerve regeneration strategies. PMID:23557939

  1. Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Wendy C Carcamo

    Full Text Available Cytoplasmic filamentous rods and rings (RR structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.Distinct cytoplasmic rods (∼3-10 µm in length and rings (∼2-5 µm in diameter in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1 and inosine monophosphate dehydrogenase 2 (IMPDH2 were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%; upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

  2. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

    International Nuclear Information System (INIS)

    Xia, Xi; Weng, Yanjie; Liao, Shujie; Han, Zhiqiang; Liu, Ronghua; Zhu, Tao; Wang, Shixuan; Xu, Gang; Meng, Li; Zhou, Jianfeng; Ma, Ding; Ma, Quanfu; Li, Xiao; Ji, Teng; Chen, Pingbo; Xu, Hongbin; Li, Kezhen; Fang, Yong; Weng, Danhui

    2011-01-01

    P21 (WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment

  3. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    International Nuclear Information System (INIS)

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

    2014-01-01

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders

  4. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kyoung Ho [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Troy, Frederic A., E-mail: fatroy@ucdavis.edu [Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, CA 95616 (United States); Xiamen University, School of Medicine, Xiamen City (China)

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  5. Cytoplasmic streaming velocity as a plant size determinant.

    Science.gov (United States)

    Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

    2013-11-11

    Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

    Science.gov (United States)

    Christie, Joshua R; Beekman, Madeleine

    2017-03-01

    Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Urinary CD4+ Effector Memory T Cells Reflect Renal Disease Activity in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

    NARCIS (Netherlands)

    Abdulahad, Wayel H.; Kallenberg, Cees G. M.; Limburg, Pieter C.; Stegeman, Coen A.

    Objective. Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration

  8. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    International Nuclear Information System (INIS)

    Ruel, Nancy; Zago, Anna; Spear, Patricia G.

    2006-01-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity

  9. Magnetite nanoparticles as reporters for microcarrier processing in cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Reibetanz, Uta, E-mail: uta.reibetanz@medizin.uni-leipzig.de [Translational Centre for Regenerative Medicine (TRM) Leipzig, Universitaet Leipzig, Philipp-Rosenthal-Strasse 55, 04103 Leipzig (Germany); Institute for Medical Physics and Biophysics, Medical Faculty, Universitaet Leipzig, Haertelstrasse 16-18, 04107 Leipzig (Germany); Jankuhn, Steffen, E-mail: jankuhn@uni-leipzig.de [Division of Nuclear Solid State Physics, Faculty of Physics and Geosciences, Universitaet Leipzig, Linnestrasse 5, 04103 Leipzig (Germany); Office for Environmental Protection and Occupational Safety, Universitaet Leipzig, Ritterstrasse 24, 04109 Leipzig (Germany)

    2011-10-15

    The development and therapeutic application of drug delivery systems based on colloidal microcarriers layer-by-layer coated with biopolyelectrolytes requires the investigation of their processing inside the cell for the successful and efficient transport and release of the active agents. The present study is focused on the time-dependent multilayer decomposition and the subsequent release of active agents to the cytoplasm. Magnetite nanoparticles (MNP) were used as reporter agents integrated into the protamine sulfate/dextran sulfate basis multilayer on colloidal SiO{sub 2} cores. This functionalization allows the monitoring of the multilayer decomposition due to the detection of the MNP release, visualized by means of proton-induced X-ray emission (PIXE) by elemental distribution of Si and Fe. The direct correlation between the microcarrier localization in endolysosomes and cytoplasm of HEK293T/17 cells via confocal laser scanning microscopy (CLSM) and the elemental distribution (PIXE) allows tracing the fate of the MNP-coated microcarriers in cytoplasm, and thus the processing of the multilayer. Microcarrier/cell co-incubation experiments of 6 h, 24 h, 48 h, and 72 h show that a MNP release and a slight expansion into the cytoplasm occurs after a longer co-incubation of 72 h.

  10. Cultivo de células de Schwann, un modelo del microambiente del sistema nervioso

    Directory of Open Access Journals (Sweden)

    Vilma C. Muñetón

    1998-03-01

    Full Text Available Algunos aspectos de la fisiopatología del sistema nervioso periférico pueden ser ampliamente estudiados en un modelo celular in vitro, enriquecido en células de Schwann. La célula de Schwann como glía del sistema nervioso periférico produce la mielina responsable de la transmisión saltatoria del impulso, influye en la actividad neuronal y da soporte y protección axonal. A su vez es blanco de procesos que alteran la normalidad del sistema nervioso periférico como neuropatías congénitas y 10 desmielinizantes, lesiones nerviosas, respuesta a patógenos neurotrópicos, etc., eventos más frecuentes y discapacitantes en individuos adultos. De ahí la importancia de obtener células a partir de animales adultos. Sin embargo, estas células son mitóticamente ""lentas"" y su obtención en cultivo requiere de condiciones específicas que estimulen su proliferación y actividad. Describimos a continuación, un modelo in vitro mediante el cual se obtienen cultivos enriquecidos en células de Schwann de ratón adulto, las cuales conservan características de las células in vivo, lo cual permite estudiar diversos fenómenos específicos del sistema nervioso periférico.

  11. Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

    Directory of Open Access Journals (Sweden)

    David-Watine Brigitte

    2006-05-01

    Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

  12. Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the memb......Aim: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain...... of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell...... membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. Methods: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3...

  13. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Lihua [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Center of Molecular Medicine, School of Medicine, Hubei University of Arts and Sciences, Xiangyang 441053 (China); Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Wang, Xiong; Huselstein, Celine [Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS – Université de Lorraine, Biopôle, 54500 Vandoeuvre-lès-Nancy (France); Chen, Yun, E-mail: yunchen@whu.edu.cn [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  14. A Novel Growth-Promoting Pathway Formed by GDNF-Overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse formation, and Partial Recovery of Function after Spinal Cord Injury

    Science.gov (United States)

    Deng, Lingxiao; Deng, Ping; Ruan, Yiwen; Xu, Zao Cheng; Liu, Naikui; Wen, Xuejun; Smith, George M.; Xu, Xiao-Ming

    2013-01-01

    Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells (SCs) overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function. PMID:23536080

  15. Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells

    International Nuclear Information System (INIS)

    Duncan, L; Shelmerdine, H; Hughes, M P; Coley, H M; Huebner, Y; Labeed, F H

    2008-01-01

    Dielectrophoresis (DEP)-the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K + and Ca 2+ levels were extremely similar between sensitive and resistant lines, levels of Cl - were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function. (note)

  16. Inhibitiory properties of cytoplasmic extract of Lactobacilli isolated from common carp intestine on human chronic myelocytic leukemia K562 cell line: an in vitro study

    Directory of Open Access Journals (Sweden)

    Kabiri F

    2011-03-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Lactobacillus species are genetically diverse groups of Lactic Acid Bacteria (LAB that have been introduced as probiotics, because of some characteristics such as their anti-tumor properties, helping the intestinal flora balance, production of antibiotics, stimulation of host immune response, etc. The aim of this study was to investigate the effects of cytoplasmic extraction and cell wall of Lactobacillus species isolated from the intestine of common carp on human chronic myelocytic leukemia or K562 cancer cell lines."n"nMethods: The intestinal contents of 115 common carp captured from the natural resources of West Azerbaijan province in Iran were examined for LAB. After isolation, the identification of Lactobacilli was done according to traditional and molecular bacteriological tests. Subsequently, a suspension of each bacterium was prepared and the protein content of the cytoplasm was extracted. Cell wall disintegration was done by cell lysis buffer and sonication. The effects of cytoplasmic extraction and cell wall on K562 cell line proliferation were investigated by MTT assays."n"nResults: The cytoplasmic extraction of the isolated Lactobacilli had significant (p<0.05 anti

  17. Schwann cell transplantation improves reticulospinal axon growth and forelimb strength after severe cervical spinal cord contusion.

    Science.gov (United States)

    Schaal, S M; Kitay, B M; Cho, K S; Lo, T P; Barakat, D J; Marcillo, A E; Sanchez, A R; Andrade, C M; Pearse, D D

    2007-01-01

    Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting

  18. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  19. Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions.

    Science.gov (United States)

    Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

    2005-01-01

    Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.

  20. α-Synuclein pathology in the cranial and spinal nerves in Lewy body disease.

    Science.gov (United States)

    Nakamura, Keiko; Mori, Fumiaki; Tanji, Kunikazu; Miki, Yasuo; Toyoshima, Yasuko; Kakita, Akiyoshi; Takahashi, Hitoshi; Yamada, Masahito; Wakabayashi, Koichi

    2016-06-01

    Accumulation of phosphorylated α-synuclein in neurons and glial cells is a histological hallmark of Lewy body disease (LBD) and multiple system atrophy (MSA). Recently, filamentous aggregations of phosphorylated α-synuclein have been reported in the cytoplasm of Schwann cells, but not in axons, in the peripheral nervous system in MSA, mainly in the cranial and spinal nerve roots. Here we conducted an immunohistochemical investigation of the cranial and spinal nerves and dorsal root ganglia of patients with LBD. Lewy axons were found in the oculomotor, trigeminal and glossopharyngeal-vagus nerves, but not in the hypoglossal nerve. The glossopharyngeal-vagus nerves were most frequently affected, with involvement in all of 20 subjects. In the spinal nerve roots, Lewy axons were found in all of the cases examined. Lewy axons in the anterior nerves were more frequent and numerous in the thoracic and sacral segments than in the cervical and lumbar segments. On the other hand, axonal lesions in the posterior spinal nerve roots appeared to increase along a cervical-to-sacral gradient. Although Schwann cell cytoplasmic inclusions were found in the spinal nerves, they were only minimal. In the dorsal root ganglia, axonal lesions were seldom evident. These findings indicate that α-synuclein pathology in the peripheral nerves is axonal-predominant in LBD, whereas it is restricted to glial cells in MSA. © 2015 Japanese Society of Neuropathology.

  1. Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

    2015-04-03

    Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

  2. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum.

    Science.gov (United States)

    Lee, Jung-Woo; Shin, Jung-Gul; Kim, Eun Hee; Kang, Hae Eun; Yim, In Been; Kim, Ji Yeon; Joo, Hong-Gu; Woo, Hee Jong

    2004-03-01

    The immunomodulatory and antitumor effects of lactic acid bacteria (LABs) were investigated. Cytoplasmic fraction of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium longum were tested for the antiproliferative activity in vitro to SNUC2A, SNU1, NIH/3T3 and Jurkat cell lines by crystal violet assay. All cytoplasmic fraction suppressed proliferation of tumor cells, though L. casei and B. longum were more effective. From these results, cytoplasmic fraction of L. casei and B. longum with Y400 as a control were administered as dietary supplements to Balb/c mice for 2, and 4 consecutive wks. Administration for 4 wks enhanced the number of total T cells, NK cells and MHC class II+ cells, and CD4-CD8+ T cells in flow cytometry analysis. To determine of antitumor activity of LABs preparation in vivo, F9 teratocarcinoma cells were inoculated on mice at 14th day. Body weight was decreased with increased survival rate in all groups with the cytoplasm of LABs. Our results showed that cytoplasmic fraction of LABs had direct antiproliferative effects on tumor cell lines in vitro, effects on immune cells in vivo, and antitumor effects on tumor-bearing mice with prolonged survival periods.

  3. Development of cardiac parasympathetic neurons, glial cells, and regional cholinergic innervation of the mouse heart.

    Science.gov (United States)

    Fregoso, S P; Hoover, D B

    2012-09-27

    Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100β calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous

  4. Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

    Science.gov (United States)

    Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

    2008-11-01

    Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

  5. Expression of Anion Exchanger 1 Sequestrates p16 in the Cytoplasm in Gastric, Colonic Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Wei-Wei Shen

    2007-10-01

    Full Text Available p16INK4A (p16 binds to cyclin-dependent kinase 4/6, negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AEi in the cytoplasm in poorly, moderately differentiated gastric, colonic adenocarcinoma cells, in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16, AEi were not detectable. Forced expression of AEi in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AEi in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death, growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric, colonic cancers, we found that 83.33% of gastric cancers, 56.52% of colonic cancers coexpressed AEi, p16 in the cytoplasm. We conclude that AEi plays a crucial role in the pathogenesis of gastric, colonic adenocarcinoma, that p16 dysfunction is a novel pathway of carcinogenesis.

  6. Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

    Science.gov (United States)

    Itabashi, Etsuko; Kazama, Tomohiko; Toriyama, Kinya

    2009-02-01

    Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.

  7. Consequences of cytoplasmic irradiation. Studies from microbeam

    International Nuclear Information System (INIS)

    Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

    2009-01-01

    The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. (author)

  8. Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

    Science.gov (United States)

    Heude, M; Chanet, R

    1975-04-01

    The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

  9. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1975-12-01

    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  10. Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Sharmistha Chakraborty

    2013-08-01

    Full Text Available Tumor necrosis factor receptor 1 (TNFR1-associated death domain protein (TRADD is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM, the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs. PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.

  11. The Effect of Erythropoietin on S100 Protein Expression in Cochlea After Acoustic Overstimulation: An Experimental Study

    Directory of Open Access Journals (Sweden)

    Gulsen Gurgen

    2014-03-01

    Full Text Available Aim: To investigate the effect of Erythropoietin on acoustically overstimulated rat spiral ganglion neurons (SGNs using S100 protein immunostaining.Material and Method: Twenty-two Wistar albino rats were divided into three groups: healthy control group (n=7, Saline solution (n=7 and Erythropoietin injection groups (n=8. Saline solution and Erythropoietin injection groups received white noise (100 dB SPL for 3 hours. Cochlear sections were stained by silver staining technique and immunostained by S100 antibody. Results: Histochemical analysis of silver staining sections revealed normal structure and a weak staining in SGNs of healthy control group. However, dark-black cytoplasmic staining, cellular shrinkage and degeneration were detected in saline injection group. On the other hand, a few weakly stained neurons were observed in erythropoietin injection group. S100 staining demonstrated strong reaction in Schwann cells and myelin sheaths of SGNs in healthy control group (p<0.05. In saline solution injection group, Schwann cells showed moderate S100 reaction and other regions of SGNs showed weak reaction (p<0.05. In erythropoietin injection group, strong S100 expression almost similar to the healthy control group was determined, although there was an occasional decrease. Discussion: Erythropoetin may prevent noise induced SGN degeneration via protecting the Schwann cells in rat cochlea.

  12. Experimental Analysis of Cell Function Using Cytoplasmic Streaming

    Science.gov (United States)

    Janssens, Peter; Waldhuber, Megan

    2012-01-01

    This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

  13. Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

    Science.gov (United States)

    Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

    2014-09-01

    Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

  14. Cytoplasmic lipid bodies of human neutrophilic leukocytes

    International Nuclear Information System (INIS)

    Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

    1989-01-01

    The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

  15. Promising SINEs for embargoing nuclear-cytoplasmic export as an anticancer strategy.

    Science.gov (United States)

    Tan, David S P; Bedard, Philippe L; Kuruvilla, John; Siu, Lillian L; Razak, Albiruni R Abdul

    2014-05-01

    In cancer cells, the nuclear-cytoplasmic transport machinery is frequently disrupted, resulting in mislocalization and loss of function for many key regulatory proteins. In this review, the mechanisms by which tumor cells co-opt the nuclear transport machinery to facilitate carcinogenesis, cell survival, drug resistance, and tumor progression will be elucidated, with a particular focus on the role of the nuclear-cytoplasmic export protein. The recent development of a new generation of selective inhibitors of nuclear export (XPO1 antagonists) and how these novel anticancer drugs may bring us closer to the implementation of this therapeutic strategy in the clinic will be discussed.

  16. Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

    Science.gov (United States)

    Tai, Andrew W.; Chuang, Jen-Zen; Sung, Ching-Hwa

    2001-01-01

    Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia. PMID:11425878

  17. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    International Nuclear Information System (INIS)

    Patheja, Pooja; Sahu, Khageswar

    2017-01-01

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  18. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  19. High ASMA+ Fibroblasts and Low Cytoplasmic HMGB1+ Breast Cancer Cells Predict Poor Prognosis.

    Science.gov (United States)

    Amornsupak, Kamolporn; Jamjuntra, Pranisa; Warnnissorn, Malee; O-Charoenrat, Pornchai; Sa-Nguanraksa, Doonyapat; Thuwajit, Peti; Eccles, Suzanne A; Thuwajit, Chanitra

    2017-10-01

    The influence of cancer-associated fibroblasts (CAFs) and high mobility group box 1 (HMGB1) has been recognized in several cancers, although their roles in breast cancer are unclear. The present study aimed to determine the levels and prognostic significance of α-smooth muscle actin-positive (ASMA + ) CAFs, plus HMGB1 and receptor for advanced glycation end products (RAGE) in cancer cells. A total of 127 breast samples, including 96 malignant and 31 benign, were examined for ASMA, HMGB1, and RAGE by immunohistochemistry. The χ 2 test and Fisher's exact test were used to test the association of each protein with clinicopathologic parameters. The Kaplan-Meier method or log-rank test and Cox regression were used for survival analysis. ASMA + fibroblast infiltration was significantly increased in the tumor stroma compared with that in benign breast tissue. The levels of cytoplasmic HMGB1 and RAGE were significantly greater in the breast cancer tissue than in the benign breast tissues. High ASMA expression correlated significantly with large tumor size, clinical stage III-IV, and angiolymphatic and perinodal invasion. In contrast, increased cytoplasmic HMGB1 correlated significantly with small tumor size, pT stage, early clinical stage, luminal subtype (but not triple-negative subtype), and estrogen receptor and progesterone receptor expression. The levels of ASMA (hazard ratio, 14.162; P = .010) and tumor cytoplasmic HMGB1 (hazard ratio, 0.221; P = .005) could serve as independent prognostic markers for metastatic relapse in breast cancer patients. The ASMA-high/HMGB1-low profile provided the most reliable prediction of metastatic relapse. We present for the first time, to the best of our knowledge, the potential clinical implications of the combined assessment of ASMA + fibroblasts and cytoplasmic HMGB1 in breast cancer. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    International Nuclear Information System (INIS)

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

  1. Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

    Institute of Scientific and Technical Information of China (English)

    Amy L Samuels; Alison Louw; Reza Zareie; Evan Ingley

    2017-01-01

    AIM To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54.METHODS HEK293 T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate(PMA). Cells were transfected with eG FP-tagged Ankrd54 with or without Lyn tyrosine kinase(wild-type, Y397 F mutant, or Y508 F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagT M gel retardation. Phosphorylated peptides were analysed by multiplereaction-monitoring(MRM) proteomic analysis.RESULTS Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tag TM gel retardation. Co-expression of an active form of the PKCδisoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54(Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased coimmunoprecipitation and enhanced co-localization in the cytoplasm.CONCLUSION We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

  2. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Funasaka, Tatsuyoshi, E-mail: funasaka@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Balan, Vitaly; Raz, Avraham [Department of Oncology and Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI (United States); Wong, Richard W., E-mail: rwong@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Bio-AFM Frontier Research Center, Kanazawa Kanazawa University, Ishikawa (Japan)

    2013-04-26

    Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

  3. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    International Nuclear Information System (INIS)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

    2009-01-01

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  4. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China); Sun, Xiaofang, E-mail: xiaofangsun@hotmail.com [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China)

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  5. Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.

    Science.gov (United States)

    Niwayama, Ritsuya; Nagao, Hiromichi; Kitajima, Tomoya S; Hufnagel, Lars; Shinohara, Kyosuke; Higuchi, Tomoyuki; Ishikawa, Takuji; Kimura, Akatsuki

    2016-01-01

    Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

  6. Kynurenine aminotransferase in the supratentorial dura mater of the rat: effect of stimulation of the trigeminal ganglion.

    Science.gov (United States)

    Knyihár-Csillik, Elizabeth; Chadaide, Zoltán; Okuno, Etsuo; Krisztin-Péva, Beata; Toldi, József; Varga, Csaba; Molnár, Andor; Csillik, Bert; Vécsei, László

    2004-04-01

    Electrical stimulation of the trigeminal ganglion has been widely used as a model of nociception, characterizing migraine. This treatment is known to evoke release of neuropeptides and neurotransmitters from nerve fibers of the dura mater. On the basis of immunocytochemical investigations, we found that under normal conditions, surface membranes of Schwann cells surrounding nerve fibers in the supratentorial dura mater display kynurenine aminotransferase-immunoreaction (KAT-IR); also KAT-IR are the granules of mast cells and the cytoplasms of macrophages (histiocytes). In consequence of stimulation of the trigeminal ganglion, Schwann cells in the dura mater became conspicuously swollen while their KAT-IR decreased considerably; also KAT-IR of mast cells and macrophages decreased significantly. At the same time, nitric oxide synthase (NOS)-IR of nerve fibers in the dura mater increased, suggesting release of nitric oxide (NO), this is known to be involved in NMDA receptor activation leading to vasodilation followed by neurogenic inflammation. Because kynurenic acid (KYNA) is an antagonist of NMDA receptors, we hypothesize that KYNA and its synthesizing enzyme, KAT, may play a role in the prevention of migraine attacks.

  7. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

    Science.gov (United States)

    Patheja, Pooja; Sahu, Khageswar

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

    Science.gov (United States)

    Belloncik, S; Chagnon, A

    1980-01-01

    A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

  9. A new sodium channel {alpha}-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2

    Energy Technology Data Exchange (ETDEWEB)

    Beckers, M.C.; Ernst, E.; Gros, P. [McGill Univ., Montreal (Canada)

    1996-08-15

    We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an {alpha}-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel {alpha}-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2. 17 refs., 1 fig., 3 tabs.

  10. TRIM5α association with cytoplasmic bodies is not required for antiretroviral activity

    International Nuclear Information System (INIS)

    Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph

    2005-01-01

    The tripartite motif (TRIM) protein, TRIM5α, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5α. In addition to diffuse cytoplasmic staining, the TRIM5α proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5α from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5α proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5α-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5α fusion proteins indicated that no TRIM5α domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5α

  11. Contribution of the actomyosin motor to the temperature-dependent translational diffusion of water by cytoplasmic streaming in Elodea canadensis cells.

    Science.gov (United States)

    Vorob'ev, V N; Anisimov, A V; Dautova, N R

    2004-12-01

    The extent to which the actomyosin motor responsible for cytoplasmic streaming contributes to the translational diffusion of water in Elodea canadensis cells was studied by a nuclear magnetic resonance (NMR) spin-echo technique. The relative contribution of the actomyosin motor was determined from the corresponding apparent diffusion coefficient by the Einstein-Smolukhovsky relation. It is equal to the difference between the diffusional displacements of the cytoplasmic and the bulk water (deltaX). The NMR data show that the temperature dependence of deltaX is humpshaped, which is characteristic of enzyme reactions. At the same time, the apparent diffusion coefficient of cytoplasmic water increases with an increase in temperature. The most significant contribution of the actomyosin motor to deltaX is observed at temperatures below 20 degrees C. Within the temperature range of 20 to 33 degrees C, deltaX changes only slightly, and a further increase in temperature reduces deltaX to zero.

  12. Polyurethane/Gelatin Nanofibrils Neural Guidance Conduit Containing Platelet-Rich Plasma and Melatonin for Transplantation of Schwann Cells.

    Science.gov (United States)

    Salehi, Majid; Naseri-Nosar, Mahdi; Ebrahimi-Barough, Somayeh; Nourani, Mohammdreza; Khojasteh, Arash; Farzamfar, Saeed; Mansouri, Korosh; Ai, Jafar

    2018-04-01

    The current study aimed to enhance the efficacy of peripheral nerve regeneration using a biodegradable porous neural guidance conduit as a carrier to transplant allogeneic Schwann cells (SCs). The conduit was prepared from polyurethane (PU) and gelatin nanofibrils (GNFs) using thermally induced phase separation technique and filled with melatonin (MLT) and platelet-rich plasma (PRP). The prepared conduit had the porosity of 87.17 ± 1.89%, the contact angle of 78.17 ± 5.30° and the ultimate tensile strength and Young's modulus of 5.40 ± 0.98 MPa and 3.13 ± 0.65 GPa, respectively. The conduit lost about 14% of its weight after 60 days in distilled water. The produced conduit enhanced the proliferation of SCs demonstrated by a tetrazolium salt-based assay. For functional analysis, the conduit was seeded with 1.50 × 10 4 SCs (PU/GNFs/PRP/MLT/SCs) and implanted into a 10-mm sciatic nerve defect of Wistar rat. Three control groups were used: (1) PU/GNFs/SCs, (2) PU/GNFs/PRP/SCs, and (3) Autograft. The results of sciatic functional index, hot plate latency, compound muscle action potential amplitude and latency, weight-loss percentage of wet gastrocnemius muscle and histopathological examination using hematoxylin-eosin and Luxol fast blue staining, demonstrated that using the PU/GNFs/PRP/MLT conduit to transplant SCs to the sciatic nerve defect resulted in a higher regenerative outcome than the PU/GNFs and PU/GNFs/PRP conduits.

  13. Infection of human and non-human cells by a highly fusogenic primary CD4-independent HIV-1 isolate with a truncated envelope cytoplasmic tail

    International Nuclear Information System (INIS)

    Saha, Kunal; Yan Hui; Nelson, Julie A.E.; Zerhouni-Layachi, Bouchra

    2005-01-01

    Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis

  14. Species-specific control of cellular proliferation and the impact of large animal models for the use of olfactory ensheathing cells and Schwann cells in spinal cord repair.

    Science.gov (United States)

    Wewetzer, Konstantin; Radtke, Christine; Kocsis, Jeffery; Baumgärtner, Wolfgang

    2011-05-01

    Autologous transplantation of olfactory ensheathing cells (OECs) and Schwann cells (SCs) is considered a promising option to promote axonal regrowth and remyelination after spinal cord injury in humans. However, if the experimental data from the rodent model can be directly extrapolated to humans, as widely believed, remains to be established. While limitations of the rodent system have recently been discussed with regard to the distinct organization of the motor systems, the question whether OECs and SCs may display species-specific properties has not been fully addressed. Prompted by recent studies on canine and porcine glia, we performed a detailed analysis of the in vitro and in vivo properties of OECs and SCs and show that rodent but not human, monkey, porcine, and canine glia require mitogens for in vitro expansion, display a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen stimulation. These data indicate fundamental inter-species differences of the control of cellular proliferation. Whether OECs and SCs from large animals and humans share growth-promoting in vivo properties with their rodent counterpart is not yet clear. Autologous implantation studies in humans did not reveal adverse effects of cell transplantation so far. However, in vivo studies of large animal or human glia and rodent recipients mainly focused on the remyelinating potential of the transplanted cells. Thus, further experimental in vivo studies in large animals are essential to fully define the axonal growth-promoting potential of OECs and SCs. Based on the homology of the in vitro growth control between porcine, canine and human glia, it is concluded that these species may serve as valuable translational models for scaling up human procedures. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair. Copyright © 2010 Elsevier Inc. All rights

  15. The effect of ionizing irradiation on motion of cytoplasm in cells of Elodea canadensis

    International Nuclear Information System (INIS)

    Tordyiya, N.V.; Grodzyins'kij, D.M.; Danil'chenko, O.O.

    1999-01-01

    The effect of acute irradiation on the velocity of cytoplasm is investigated. It is shown that, for small doses, there is a strong nonlinearity between the velocity of cytoplasm and dose. The nonlinear behavior disappears with increasing a dose

  16. Interferometric measurements of dry mass content in nuclei and cytoplasm in the life cycle of antheridial filaments cells of Chara vulgaris L. in their successive developmental stages

    Directory of Open Access Journals (Sweden)

    Hanna Kuran

    2015-01-01

    Full Text Available Interferometric measurements of the nucleus and cytoplasm dry mass during interphase in the successive stages of development of antheridial filaments of Chara vulgaris demonstrated that the dry mass and surface area of cell nuclei double in size in each of the successive generations of the filaments, whereas neither the surface nor the dry mass of the cytoplasm increase in such proportion in the same period. In the successive stages of development of the antheridial filaments the dry mass and surface area of the nuclei and cytoplasm gradually diminish.

  17. Combining neurotrophin-transduced schwann cells and rolipram to promote functional recovery from subacute spinal cord injury.

    Science.gov (United States)

    Flora, Govinder; Joseph, Gravil; Patel, Samik; Singh, Amanpreet; Bleicher, Drew; Barakat, David J; Louro, Jack; Fenton, Stephanie; Garg, Maneesh; Bunge, Mary Bartlett; Pearse, Damien D

    2013-01-01

    Following spinal cord injury (SCI), both an inhibitory environment and lack of intrinsic growth capacity impede axonal regeneration. In a previous study, prevention of cyclic adenosine monophosphate (AMP) hydrolysis by the phosphodiesterase-4 inhibitor rolipram, in combination with Schwann cell (SC) grafts, promoted significant supraspinal and proprioceptive fiber growth and/or sparing and improved locomotion. In another study, transplanted SCs transduced to generate a bifunctional neurotrophin (D15A) led to significant increases in graft SCs and axons, including supraspinal and myelinated axons. Here we studied the growth and myelination of local and supraspinal axons and functional outcome following the combination of rolipram administration and neurotrophin-transduced SC implantation after SCI. Rolipram was administered subcutaneously for 4 weeks immediately after contusion at vertebral T8 (25.0-mm weight drop, MASCIS impactor). GFP or GFP-D15A-transduced SCs were injected into the injury epicenter 1 week after SCI. GFP-D15A SC grafts and GFP SC grafts with rolipram contained significantly more serotonergic fibers compared to GFP SCs. SC myelinated axons were increased significantly in GFP SC with rolipram-treated animals compared to animals receiving SCI alone. Rolipram administered with either GFP or GFP-D15A SCs significantly increased numbers of brain stem-derived axons below the lesion/implant area and improved hindlimb function. Compared to the single treatments, the combination led to the largest SC grafts, the highest numbers of serotonergic fibers in the grafts, and increased numbers of axons from the reticular formation below the lesion/implant area and provided the greatest improvement in hindlimb function. These findings demonstrate the therapeutic potential for a combination therapy involving the maintenance of cyclic AMP levels and neurotrophin-transduced SCs to repair the subacutely injured spinal cord.

  18. Coulomb interactions between cytoplasmic electric fields and phosphorylated messenger proteins optimize information flow in cells.

    Directory of Open Access Journals (Sweden)

    Robert A Gatenby

    2010-08-01

    Full Text Available Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM. While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl- and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length.Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions.This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger

  19. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

    2010-08-06

    The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

  20. Sugar Composition Analysis of Fuzi Polysaccharides by HPLC-MSn and Their Protective Effects on Schwann Cells Exposed to High Glucose

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    Bei-Bei Wang

    2016-11-01

    Full Text Available Fuzi has been used to treat diabetic complications for many years in china. In a previous study, we have shown that Fuzi aqueous extract can attenuate Diabetic peripheral neuropathy (DPN in rats and protect Schwann cells from injury. Thus, the protective effect of Fuzi polysaccharides (FPS on high glucose-induced SCs and the preliminary mechanism were investigated. Firstly, the FPS were obtained and their monose composition was analyzed by the combination of pre-column derivatization and high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry (HPLC/ESI-MSn. The results witnessed the efficiency of this method and seven monosaccharides were tentatively identified, among which fucose was first reported. Simultaneously, m/z 215 can be considered as diagnostic ions to confirm the number of monosaccharides. Next, high glucose-induced SC model was applied and divided into model group, treated group of FPS, normal and osmotic control group. After treatment for 48 h, the data showed FPS could significantly decrease the intracellular ROS and apoptosis, which were determined by the corresponding fluorescent probes. Then, the expression of oxidative stress-related proteins in SCs were measured by Western blot. Furthermore, the protein tests found that FPS markedly up-regulated superoxide dismutase (SOD, catalase (CAT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α protein level, but down-regulated NADPH oxidase-1 (Nox1 protein level. Moreover, FPS could also increase AMP-activated protein kinase (AMPK activation significantly. Hence, we preliminary deduced that AMPK-PGC-1α pathway may play an important role in the protective effect of FPS against high glucose-induced cell damage.

  1. Genetic expression of induced rice sterility under alien-cytoplasm

    International Nuclear Information System (INIS)

    Wang Naiyuan; Cai Zhijun; Liang Kangjing; Li Yu

    2005-01-01

    Rice restorer lines were treated with 60 Co γ-ray and 4 male sterile mutants obtained with the fertility of controlled by 4 non-allelic recessive genes, respectively. Sixty combinations were made by using male sterile plants/fertile plants as male parents, and 15 different cytoplasmic substitution lines of the same cell nucleus as female parents. The result showed that F 1 spikelets were normal and fertile, and different numbers of male sterile plants were segregated in F 2 . Complete fertility genotype was not found among interactions between induced male sterile genes and alien-cytoplasms. (authors)

  2. Raman microspectroscopy of nucleus and cytoplasm for human colon cancer diagnosis.

    Science.gov (United States)

    Liu, Wenjing; Wang, Hongbo; Du, Jingjing; Jing, Chuanyong

    2017-11-15

    Subcellular Raman analysis is a promising clinic tool for cancer diagnosis, but constrained by the difficulty of deciphering subcellular spectra in actual human tissues. We report a label-free subcellular Raman analysis for use in cancer diagnosis that integrates subcellular signature spectra by subtracting cytoplasm from nucleus spectra (Nuc.-Cyt.) with a partial least squares-discriminant analysis (PLS-DA) model. Raman mapping with the classical least-squares (CLS) model allowed direct visualization of the distribution of the cytoplasm and nucleus. The PLS-DA model was employed to evaluate the diagnostic performance of five types of spectral datasets, including non-selective, nucleus, cytoplasm, ratio of nucleus to cytoplasm (Nuc./Cyt.), and nucleus minus cytoplasm (Nuc.-Cyt.), resulting in diagnostic sensitivity of 88.3%, 84.0%, 98.4%, 84.5%, and 98.9%, respectively. Discriminating between normal and cancerous cells of actual human tissues through subcellular Raman markers is feasible, especially when using the nucleus-cytoplasm difference spectra. The subcellular Raman approach had good stability, and had excellent diagnostic performance for rectal as well as colon tissues. The insights gained from this study shed new light on the general applicability of subcellular Raman analysis in clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

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    Aleksandra Glogowska

    2012-05-01

    Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

  4. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Directory of Open Access Journals (Sweden)

    Q. Sun

    2012-10-01

    Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  5. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    International Nuclear Information System (INIS)

    Sun, Q.; Xiong, J.; Lu, J.; Xu, S.; Li, Y.; Zhong, X.P.; Gao, G.K.; Liu, H.Q.

    2012-01-01

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy

  6. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  7. The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.

    Science.gov (United States)

    Isern, Joan; García-García, Andrés; Martín, Ana M; Arranz, Lorena; Martín-Pérez, Daniel; Torroja, Carlos; Sánchez-Cabo, Fátima; Méndez-Ferrer, Simón

    2014-09-25

    Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.

  8. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  9. Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

    Directory of Open Access Journals (Sweden)

    Tentler John J

    2011-08-01

    Full Text Available Abstract Background The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. Results To map the minimal functional nuclear localization (NLS and nuclear export (NES signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247 in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112 and another to the DNA binding domain (DBD, (NES2: 275LWEFIRDILI284. Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323 by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. Conclusions These data highlight that ESE-1 contains NLS and NES signals that play a critical role in

  10. Cytoplasmic Control of Sense-Antisense mRNA Pairs

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    Flore Sinturel

    2015-09-01

    Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

  11. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    Science.gov (United States)

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Nuclear and Cytoplasmic Delivery of Lactoferrin in Glioma using Chitosan Nanoparticles: Cellular Location Dependent-Action of Lactoferrin.

    Science.gov (United States)

    Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

    2018-05-23

    Lactoferrin (Lf) exerts anti-cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti-cancer effect. Upon treatment with 300 and 800 µg/mL Lf loaded chitosan NPs, nuclear targeted Lf-NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf-NPs at 300 µg/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti-cancer protein. Copyright © 2018. Published by Elsevier B.V.

  13. Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

    Directory of Open Access Journals (Sweden)

    Mirko eZaffagnini

    2013-11-01

    Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively-modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively-modified GAPDH in stress signaling pathways in plants.

  14. Uptake of 3H-thymidine by the receptor cell populations after injury of the sensory nerve fibres

    International Nuclear Information System (INIS)

    Chuchkov, Ch.N.

    1978-01-01

    The material of the study was the skin from the beak of two-day ducklings. The investigation was carried out on the 2nd, 5th, 20th and 45th day after the crushing of the sensory nerve fibres entering the capsulated Herbst receptors. Twenty four hours before the biopsy, the ducklings were injected at 6 hours intervals with 3 H-thymidine. The number of labelled index in the three cell pupulations, participating in the receptor development was determined. The cells of the subcapsular space of all control animals (with intacted suborbital nerves) have shown the highest labelled index. The index of the capsular perineural cells is about 12 times lower, while the labelled index of the Schwann receptor cells is about 10 times lower. Following the denervation, the labelled index in increasing and reaches its maximum on the 5th postoperative day. The Schwann receptor cells in comparison to the two other cell populations show the most significant deviation during the regeneration (45th day after the intervention). The investigations show that all three cell populations pass through a miotic cycle of innovation. The low labelled index of the Schwann receptors (1-2 labelled cells in 1000) is an indication of a high differentiation. One can assume that their regeneration takes place at the expense of the proper proliferation activity as well as of the differentiation of the Schwann cells from the distal section of the regenerating sensory nerve fibres. Taking into consideration the high labelled index of the other populations, it seems most probable that their regeneration takes place for the expense of their own cell populations. (A.B.)

  15. Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1976-01-01

    The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  16. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    Science.gov (United States)

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-05-01

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

  17. Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

    Science.gov (United States)

    Valkenburg, J A; Woldringh, C L

    1984-01-01

    The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid. Images PMID:6389508

  18. DNA precursor compartmentation in mammalian cells: distribution and rates of equilibration between nucleus and cytoplasm

    International Nuclear Information System (INIS)

    Leeds, J.M.

    1986-01-01

    A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Experiments comparing the labeling kinetics of 3 H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of 3 H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells

  19. Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

    Science.gov (United States)

    Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

    2018-01-15

    Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in

  20. Labelling of human resting lymphocytes by continuous infusion of (/sup 3/H)thymidine. 1. Characterization of cytoplasmic label

    Energy Technology Data Exchange (ETDEWEB)

    Schick, P; Trepel, F; Maisel, K H; Past, W; Reisert, I; Begemann, H; Pilgrim, C [Ulm Univ. (Germany, F.R.)

    1978-01-01

    After continuous /sup 3/H-TdR infusion in vivo or incubation with /sup 3/H-TdR in vitro human blood lymphocytes were examined by light-microscopic and electron-microscopic autoradiography. Using relatively long autoradiographic exposure times (50-300 days) not only nuclear but also cytoplasmic labelling was visualized, the cytoplasmic label being present in up to 96% of the cells. The cytoplasmic label was predominantly associated with the mitochondria and was removed from the cells nearly completely by treatment with DNase but not with RNase or cold perchloric acid. It is concluded that this cytoplasmic label mainly represents /sup 3/H-TdR incorporated into mitochondrial DNA which is continuously renewed in an average turnover time of 14 days or less. This value is compatible with a turnover time of 11 days for mitochondrial DNA in mammalian cells reported in the literature.

  1. Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

    Science.gov (United States)

    Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

    2016-01-01

    Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

  2. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    International Nuclear Information System (INIS)

    Kumari, Gita; Mahalingam, S.

    2009-01-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  3. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  4. Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

    Science.gov (United States)

    Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

    2016-05-01

    Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

    International Nuclear Information System (INIS)

    Soldatenkov, V.A.; Filippovich, I.V.

    1986-01-01

    A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

  6. Glucocorticoid control of rat growth hormone gene expression: Effect on cytoplasmic messenger ribonucleic acid production and degradation

    International Nuclear Information System (INIS)

    Gertz, B.J.; Gardner, D.G.; Baxter, J.D.

    1987-01-01

    The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of [3H]uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: (1) pulse-chase studies and (2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production

  7. Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

    Science.gov (United States)

    Sander, Suzanne; Arora, Neha; Smith, Emily A

    2012-06-01

    Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

  8. PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

    Science.gov (United States)

    Mollenhauer, Hilton H.; Zebrun, William

    1960-01-01

    Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

  9. Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm.

    Science.gov (United States)

    Kyne, Ciara; Crowley, Peter B

    2017-09-19

    Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal -GR n (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR 5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

  10. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

    Science.gov (United States)

    Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

    2016-01-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  11. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Genetic analysis of the cytoplasmic dynein subunit families.

    Science.gov (United States)

    Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  13. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  14. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

    2016-01-01

    ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... checkpoints, initiating DNA repair and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

  15. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  16. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    International Nuclear Information System (INIS)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-01

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells

  17. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Lyi, Sangbom Michael; Tan, Min Jie Alvin, E-mail: tanmja@gis.a-star.edu.sg; Parrish, Colin R., E-mail: crp3@cornell.edu

    2014-05-15

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

  18. Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

    International Nuclear Information System (INIS)

    Lyi, Sangbom Michael; Tan, Min Jie Alvin; Parrish, Colin R.

    2014-01-01

    Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes

  19. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

    International Nuclear Information System (INIS)

    Kamen, B.A.; Wang, M.T.; Streckfuss, A.J.; Peryea, X.; Anderson, R.G.

    1988-01-01

    MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[ 3 H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[ 3 H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface

  20. Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

    Science.gov (United States)

    Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

    2008-07-15

    Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

  1. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    Directory of Open Access Journals (Sweden)

    Christian Much

    2016-06-01

    Full Text Available Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

  2. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    Science.gov (United States)

    Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

    2016-06-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

  3. Data on the effect of in vivo knockdown using artificial ErbB3 miRNA on Remak bundle structure

    Directory of Open Access Journals (Sweden)

    Yuki Miyamoto

    2017-06-01

    Full Text Available Mature Schwann cells, the peripheral nervous system (PNS glial cells, have two major roles for neuronal axons (Bunge, 1993 [1]. For large diameter axons, Schwann cells form myelin sheaths with multiple layers. For small diameter axons, they form Remak bundle composed only of single layer of the Schwann cell plasma membrane. In the PNS, ErbB3 forms a dimer with ErbB2 on the Schwann cell plasma membrane. ErbB3 plays a key role in myelination by myelinating Schwann cells, that is to say, its role in myelin thickness. Herein we provide the data regarding the effect of in vivo knockdown of ErbB3 on the thickness between an axon and a neighboring axon in Remak bundle, which is formed by non-myelinating Schwann cells. Since ErbB3 knockout mice are embryonically lethal, Schwann cell lineage-specific transgenic mice transcribing ErbB3 shRNA with an artificial miRNA backbone were generated and used in these experiments (Torii et al., 2014 [2].

  4. Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding.

    Directory of Open Access Journals (Sweden)

    Koh-ichi Utani

    Full Text Available Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

  5. Cytoplasmic BRMS1 expression in malignant melanoma is associated with increased disease-free survival

    Directory of Open Access Journals (Sweden)

    Slipicevic Ana

    2012-02-01

    Full Text Available Abstract Background/aims Breast cancer metastasis suppressor 1 (BRMS1 blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis. Methods Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines. Results A significantly higher percentage of nevi (87%, compared to primary melanomas (20% and metastases (48%, expressed BRMS1 in the nucelus (p Waf1/Cip1 (p = 0.009. Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013 and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033. Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016 and decreased relapse-free period (p = 0.043. Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011, a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro. Conclusion Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion. Please see related article: http://www.biomedcentral.com/1741-7015/10/19

  6. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    Administrator

    2011-04-25

    Apr 25, 2011 ... Bone marrow stromal cells (BMSCs), a type of multipotent stem cell, can differentiate into various types ... induced to differentiate into neuron-like cells when they are ... axonal regeneration and functional reconstruction do not.

  7. Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

    International Nuclear Information System (INIS)

    Sauterer, R.A.; Feeney, R.J.; Zieve, G.W.

    1988-01-01

    Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs

  8. p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

    Science.gov (United States)

    Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

    2013-03-01

    We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. A physical perspective on cytoplasmic streaming.

    Science.gov (United States)

    Goldstein, Raymond E; van de Meent, Jan-Willem

    2015-08-06

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

  10. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

    Directory of Open Access Journals (Sweden)

    Sara Marinelli

    Full Text Available In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25, the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A, could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC. We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

  11. Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

    Science.gov (United States)

    Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

    2014-01-01

    ABSTRACT Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly

  12. Tapetal-Delayed Programmed Cell Death (PCD and Oxidative Stress-Induced Male Sterility of Aegilops uniaristata Cytoplasm in Wheat

    Directory of Open Access Journals (Sweden)

    Zihan Liu

    2018-06-01

    Full Text Available Cytoplasmic male sterility (CMS plays a crucial role in the utilization of hybrid vigor. Pollen development is often accompanied by oxidative metabolism responses and tapetal programmed cell death (PCD, and deficiency in these processes could lead to male sterility. Aegilops uniaristata cytoplasmic male sterility (Mu-CMS wheat is a novel male-sterile line in wheat, which possess important potential in hybrid wheat breeding. However, its CMS mechanisms remain poorly understood. In our study, U87B1-706A, with the Aegilops uniaristata cytoplasm, and the maintainer line 706B were used to explore the abortive reason. Compared with 706B, histological analysis and PCD detection of the anther demonstrated that U87B1-706A appeared as delayed tapetal PCD as well as a disorganized organelle phenotype in the early uninucleate stage. Subsequently, a shrunken microspore and disordered exine structure were exhibited in the late uninucleate stage. While the activities of antioxidase increased markedly, the nonenzymatic antioxidant contents declined obviously following overacummulation of reactive oxygen species (ROS during pollen development in U87B1-706A. Real-time quantitative PCR testified that the transcript levels of the superoxide dismutase (SOD, catalase (CAT, and ascorbate peroxidase (APX genes, encoding pivotal antioxidant enzymes, were up-regulated in early pollen development. Therefore, we deduce excess ROS as a signal may be related to the increased expression levels of enzyme genes, thereby breaking the antioxidative system balance, resulting in delayed tapetal PCD initiation, which finally led to pollen abortion and male sterility in U87B1-706A. These results provide evidence to further explore the mechanisms of abortive pollen in CMS wheat.

  13. Entropy measures of collective cell migration

    Science.gov (United States)

    Whitby, Ariadne; Parrinello, Simona; Faisal, Aldo

    2015-03-01

    Collective cell migration is a critical process during tissue formation and repair. To this end there is a need to develop tools to quantitatively measure the dynamics of collective cell migration obtained from microscopy data. Drawing on statistical physics we use entropy of velocity fields derived from dense optic flow to quantitatively measure collective migration. Using peripheral nerve repair after injury as experimental system, we study how Schwann cells, guided by fibroblasts, migrate in cord-like structures across the cut, paving a highway for neurons. This process of emergence of organised behaviour is key for successful repair, yet the emergence of leader cells and transition from a random to ordered state is not understood. We find fibroblasts induce correlated directionality in migrating Schwann cells as measured by a decrease in the entropy of motion vector. We show our method is robust with respect to image resolution in time and space, giving a principled assessment of how various molecular mechanisms affect macroscopic features of collective cell migration. Finally, the generality of our method allows us to process both simulated cell movement and microscopic data, enabling principled fitting and comparison of in silico to in vitro. ICCS, Imperial College London & MRC Clinical Sciences Centre.

  14. Effects of chromosomal breaks induced by X-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

    International Nuclear Information System (INIS)

    Parks, L.C.; Dicker, D.T.; Conger, A.D.; Daneo-Moore, L.; Higgins, M.L.

    1981-01-01

    A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by 1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; 2) subsequent determination of the number of irradiated cells in which mesosomes and central density changes could be visualised after fixative was added. The results obtained by exposure of cells to a) doses up to 1100 krads before fixation and b) doses greater than 1100 krads before fixation suggested that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted. (author)

  15. S-layer and cytoplasmic membrane – exceptions from the typical archaeal cell wall with a focus on double membranes

    Directory of Open Access Journals (Sweden)

    Andreas eKlingl

    2014-11-01

    Full Text Available The common idea of typical cell wall architecture in archaea consists of a pseudo-crystalline proteinaceous surface layer (S-layer, situated upon the cytoplasmic membrane. This is true for the majority of described archaea, hitherto. Within the crenarchaea, the S-layer often represents the only cell wall component, but there are various exceptions from this wall architecture. Beside (glycosylated S-layers in (hyperthermophilic cren- and euryarchaea as well as halophilic archaea, one can find a great variety of other cell wall structures like proteoglycan-like S-layers (Halobacteria, glutaminylglycan (Natronococci, methanochondroitin (Methanosarcina or double layered cell walls with pseudomurein (Methanothermus and Methanopyrus. The presence of an outermost cellular membrane in the crenarchaeal species Ignicoccus hospitalis already gave indications for an outer membrane similar to Gram-negative bacteria. Although there is just limited data concerning their biochemistry and ultrastructure, recent studies on the euryarchaeal methanogen Methanomassiliicoccus luminyensis, cells of the ARMAN group, and the SM1 euryarchaeon delivered further examples for this exceptional cell envelope type consisting of two membranes.

  16. Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

    Science.gov (United States)

    Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

    2012-05-01

    Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

  17. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  18. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    International Nuclear Information System (INIS)

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae; Park, Sang Chul

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin α, karyopherin β, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  19. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  20. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    International Nuclear Information System (INIS)

    Muir, D.; Varon, S.; Manthorpe, M.

    1990-01-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens

  1. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    Energy Technology Data Exchange (ETDEWEB)

    Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  2. CONTINUOUS MEASUREMENT OF THE CYTOPLASMIC PH IN LACTOCOCCUS-LACTIS WITH A FLUORESCENT PH INDICATOR

    NARCIS (Netherlands)

    MOLENAAR, D; ABEE, T; KONINGS, WN

    1991-01-01

    The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the

  3. Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

    Directory of Open Access Journals (Sweden)

    Zhi-yuan Guo

    2015-01-01

    Full Text Available Human umbilical cord-derived mesenchymal stem cells (hUCMSCs represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

  4. Tau-mediated nuclear depletion and cytoplasmic accumulation of SFPQ in Alzheimer's and Pick's disease.

    Directory of Open Access Journals (Sweden)

    Yazi D Ke

    Full Text Available Tau dysfunction characterizes neurodegenerative diseases such as Alzheimer's disease (AD and frontotemporal lobar degeneration (FTLD. Here, we performed an unbiased SAGE (serial analysis of gene expression of differentially expressed mRNAs in the amygdala of transgenic pR5 mice that express human tau carrying the P301L mutation previously identified in familial cases of FTLD. SAGE identified 29 deregulated transcripts including Sfpq that encodes a nuclear factor implicated in the splicing and regulation of gene expression. To assess the relevance for human disease we analyzed brains from AD, Pick's disease (PiD, a form of FTLD, and control cases. Strikingly, in AD and PiD, both dementias with a tau pathology, affected brain areas showed a virtually complete nuclear depletion of SFPQ in both neurons and astrocytes, along with cytoplasmic accumulation. Accordingly, neurons harboring either AD tangles or Pick bodies were also depleted of SFPQ. Immunoblot analysis of human entorhinal cortex samples revealed reduced SFPQ levels with advanced Braak stages suggesting that the SFPQ pathology may progress together with the tau pathology in AD. To determine a causal role for tau, we stably expressed both wild-type and P301L human tau in human SH-SY5Y neuroblastoma cells, an established cell culture model of tau pathology. The cells were differentiated by two independent methods, mitomycin C-mediated cell cycle arrest or neuronal differentiation with retinoic acid. Confocal microscopy revealed that SFPQ was confined to nuclei in non-transfected wild-type cells, whereas in wild-type and P301L tau over-expressing cells, irrespective of the differentiation method, it formed aggregates in the cytoplasm, suggesting that pathogenic tau drives SFPQ pathology in post-mitotic cells. Our findings add SFPQ to a growing list of transcription factors with an altered nucleo-cytoplasmic distribution under neurodegenerative conditions.

  5. Regulation of autophagy by cytoplasmic p53.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  6. The Protective Effect of Cell Wall and Cytoplasmic Fraction of Selenium Enriched Yeast on 1, 2-Dimethylhydrazine-induced Damage in Liver

    Directory of Open Access Journals (Sweden)

    Mitra Dadrass

    2014-02-01

    Full Text Available Background: 1, 2-Dimethylhydrazine (DMH enhances lipid peroxidation rate by tumor mitochondria than normal tissue counterpart and causes many disorders in antioxidant system in liver. It also increases the level of enzymes that metabolize toxin in liver and colon. The aim of this study was to evaluate the alteration of liver and its enzymes after DMH injection and evaluate protective effect of cell wall and cytoplasmic fractions of Saccharomyces cereviseae enriched with selenium (Se on these tissues. Materials and Methods: Forty eight female rats were prepared and acclimatized to the laboratory conditions for two weeks, and all animals received 1, 2- dimethyl hydrazine chloride (40 mg/kg body weight twice a week for 4 weeks except healthy control. At first colon carcinoma (aberrant crypt foci confirmed by light microscope. Then the changes resulting from injection of DMH on liver of animals in initial and advanced stages of colon cancer were examined. In addition, the protective effect of cell wall and cytoplasmic fractions of Selenium-enriched S. cerevisiae were investigated in two phases. First phase in initial stage and second phase in advanced stage of colon cancer were performed respectively. Forty weeks following the first DMH injection, all survived animals were sacrificed. Then, colon and liver removed and exsanguinated by heart puncture. For measuring the levels of enzymes (AST, ALT, and ALP, a commercial kit (Parsazmoon, Iran and an autoanalyzer (BT 3000 Pluse, Italy were used. Results: The results showed that subcutaneous injection of DMH increased the ALT, AST, and ALP levels up to 78.5, 161.38, and 275.88 U/L compared to the control, respectively. Moreover, statistical analysis in both phases of experiment revealed that the enzyme levels were decreased in the treated groups in comparison with the DMH-injected group, while the levels of these enzymes were lower in the control group. Conclusion: It should be concluded that

  7. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

    International Nuclear Information System (INIS)

    Whitt, M.A.; Chong, L.; Rose, J.K.

    1989-01-01

    The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding

  8. Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

    Science.gov (United States)

    Mizejewski, G J

    2015-12-01

    The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer.

  9. Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

    International Nuclear Information System (INIS)

    Kim, Daeyou; Jeon, Choonju; Kim, Jeong-Hwan; Kim, Mi-Seon; Yoon, Cheol-Hee; Choi, In-Soo; Kim, Sung-Hoon; Bae, Yong-Soo

    2006-01-01

    The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused β-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-β-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material

  10. Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Woo Jin Jeong

    Full Text Available In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO can alter the function of the basement membrane of retinal pigment epithelial (RPE cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

  11. Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton.

    Science.gov (United States)

    Suzuki, Hideaki; Rodriguez-Uribe, Laura; Xu, Jiannong; Zhang, Jinfa

    2013-10-01

    A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9 %) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

  12. Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

    Science.gov (United States)

    Xia, J. H.; Roberts, JKM.

    1996-05-01

    We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

  13. Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

    NARCIS (Netherlands)

    Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  14. Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

    NARCIS (Netherlands)

    Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  15. Cytoplasmic Dynein Promotes HIV-1 Uncoating

    Directory of Open Access Journals (Sweden)

    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  16. Non-Invasive Cell-Based Therapy for Traumatic Optic Neuropathy

    Science.gov (United States)

    2013-10-01

    2004. 18(11): p. 1122-5. 4. Castillo, B., Jr., et al., Retinal ganglion cell survival is promoted by genetically modified astrocytes designed to...growth factors such as peripheral nerve ensheathing cells (Schwann cells) or cells genetically modified to release growth factors and (iv) cells with...Book: Retinal Degenerations, editors: Tombran-Tink and Barnstable. 620-2; Humana Press; Chapter 17: 317-342. Gamm DM, Wang S, Lu B, Girman S, Holmes T

  17. Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis

    NARCIS (Netherlands)

    Kallenberg, Cees G. M.

    Purpose of reviews This review focuses on recent advance in the diagnosis pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Recent findings Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and

  18. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

    Science.gov (United States)

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  19. Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

    Science.gov (United States)

    Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

    2017-01-01

    Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

  20. An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

    Science.gov (United States)

    Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

    2014-01-01

    MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

  1. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

    Science.gov (United States)

    Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2012-05-30

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation

  2. Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

    Science.gov (United States)

    Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

    2015-01-01

    During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

  3. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoud, Nora F. [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Faculty of Pharmacy, Suez Canal University, Ismailia (Egypt); Jasirwan, Chyntia [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Division of Hepatobiliary, Department of Internal Medicine, Faculty of Medicine, University of Indonesia (Indonesia); Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Nagamata, Satoshi [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe (Japan); Kawabata, Akiko [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Tang, Huamin [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Immunology, Nanjing Medical University, Nanjing (China); Mori, Yasuko, E-mail: ymori@med.kobe-u.ac.jp [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan)

    2016-03-15

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

  4. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

    International Nuclear Information System (INIS)

    Mahmoud, Nora F.; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

    2016-01-01

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

  5. Cytoplasmic Overexpression of CD95L in Esophageal Adenocarcinoma Cells Overcomes Resistance to CD95-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Gregory A. Watson

    2011-03-01

    Full Text Available Introduction: The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. Methods: CD95L expression was characterized in immortalized squamous esophagus (HET-1A and Barrett esophagus (BAR-T cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control; and KFL cells (+ control. Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. Results: Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. Conclusions: CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.

  6. Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS.

    Science.gov (United States)

    Zhang, Guigen; Chan, Baca; Samarina, Naira; Abere, Bizunesh; Weidner-Glunde, Magdalena; Buch, Anna; Pich, Andreas; Brinkmann, Melanie M; Schulz, Thomas F

    2016-02-23

    The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.

  7. The Cell Wall Teichuronic Acid Synthetase (TUAS Is an Enzyme Complex Located in the Cytoplasmic Membrane of Micrococcus luteus

    Directory of Open Access Journals (Sweden)

    Lingyi Lynn Deng

    2010-01-01

    composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6α-D-Glcp−1-]n, which is covalently anchored to the peptidoglycan on the inner cell wall and extended to the outer surface of the cell envelope. An enzyme complex responsible for the TUA chain biosynthesis was purified and characterized. The 440 kDa enzyme complex, named teichuronic acid synthetase (TUAS, is an octomer composed of two kinds of glycosyltransferases, Glucosyltransferase, and ManNAcA-transferase, which is capable of catalyzing the transfer of disaccharide glycosyl residues containing both glucose and the N-acetylmannosaminuronic acid residues. TUAS displays hydrophobic properties and is found primarily associated with the cytoplasmic membrane. The purified TUAS contains carotinoids and lipids. TUAS activity is diminished by phospholipase digestion. We propose that TUAS serves as a multitasking polysaccharide assembling station on the bacterial membrane.

  8. Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists[W][OA

    Science.gov (United States)

    Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen; Meagher, Richard B.

    2012-01-01

    Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals. PMID:22589468

  9. Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1

    International Nuclear Information System (INIS)

    Park, EunJoo; Kim, Tae-Houn

    2017-01-01

    Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1 was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction. - Highlights: • Nuclear and cytoplasmic functions of PYR1 were studied in the mutant which lacked majority of ABA responses. • Nuclear PYR1 reconstituted partially the ABA responses during seed germination, root growth, and guard cell movement. • Both the nuclear and cytoplasmic functions of PYR1 were required for the full generation of ABA responses.

  10. Peripheral neuropathy in type A Niemann-Pick disease. A morphological study.

    Science.gov (United States)

    Landrieu, P; Saïd, G

    1984-01-01

    A black boy had a severe neuropathic form of Niemann-Pick disease (NPD) with a pronounced sphingomyelinase deficiency in the fibroblasts. Nerve conduction velocities were diminished, and a nerve biopsy was performed. Isolated fibers showed segmental demyelination and numerous dense bodies in the Schwann cells (SC). Electron microscopy revealed two categories of inclusions: the first was made up of lysosomal inclusions usually described in NPD. The second comprised myelin inclusions--sometimes still connected to the original myelin sheath--indicating severe myelinopathy. Both myelin debris and NPD inclusions were found in axoplasms and probably came from SC cytoplasm through axolemma lesions. NPD is a unique example of myelinopathy due to sphingomyelinase deficiency.

  11. The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

    International Nuclear Information System (INIS)

    Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

    1989-05-01

    The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

  12. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail

    Directory of Open Access Journals (Sweden)

    Takeo eKuwata

    2013-05-01

    Full Text Available The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1 infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of nonhuman primates with simian immunodeficiency virus (SIV is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7900-fold, 1000-fold, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  13. Fine structure of subepithelial "free" and corpuscular trigeminal nerve endings in anterior hard palate of the rat.

    Science.gov (United States)

    Byers, M R; Yeh, Y

    1984-01-01

    Axonally transported protein labeled many trigeminal nerve endings in subepithelial regions of the anterior hard palate of the rat. Sensory endings were most numerous in the lamina propria near the tips of the palatal rugae where large connective tissue and epithelial papillae interdigitated. Two kinds of sensory ending were found there: "free" endings, and a variety of corpuscular endings. The "free" sensory endings consisted of bundles of unmyelinated axons separated from the connective tissue by relatively unspecialized Schwann cells covering part or all of their surface and a completely continuous basal lamina; they were commonly found running parallel to the epithelium or near corpuscular endings. The corpuscular sensory endings all had a specialized nerve form, specialized Schwann cells, and axonal fingers projecting into the corpuscular basal lamina or connective tissue. There were at least four distinct types of corpuscular ending: Ruffini-like endings were found among dense collagen bundles, and they had a flattened nerve ending with a flattened Schwann lamella on either side. Meissner endings had an ordered stack of flattened nerve terminals with flattened Schwann cells and much basal lamina within and around the corpuscle. Simple corpuscles were single nerve endings surrounded by several layers of concentric lamellar Schwann processes. Glomerular endings were found in lamina propria papillae or encircling epithelial papillae; they were a tangle of varied neural forms each of which had apposed flattened Schwann cells, and a layer of basal lamina of varied thickness. Fibroblasts often formed incomplete partitions around Meissner and simple corpuscles. The axoplasm of all kinds of subepithelial sensory endings contained numerous mitochondria and vesicles, as well as occasional multivesicular bodies and lysosomes; the axoplasm of all endings was pale with few microtubules and neurofilaments. The specialized lamellar Schwann cells had much pinocytotic

  14. Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures.

    Science.gov (United States)

    Kitiyanant, Y; Saikhun, J; Chaisalee, B; White, K L; Pavasuthipaisit, K

    2001-01-01

    Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

  15. Sciatic nerve regeneration by transplantation of Schwann cells via erythropoietin controlled-releasing polylactic acid/multiwalled carbon nanotubes/gelatin nanofibrils neural guidance conduit.

    Science.gov (United States)

    Salehi, Majid; Naseri-Nosar, Mahdi; Ebrahimi-Barough, Somayeh; Nourani, Mohammdreza; Khojasteh, Arash; Hamidieh, Amir-Ali; Amani, Amir; Farzamfar, Saeed; Ai, Jafar

    2018-05-01

    The current study aimed to enhance the efficacy of peripheral nerve regeneration using an electrically conductive biodegradable porous neural guidance conduit for transplantation of allogeneic Schwann cells (SCs). The conduit was produced from polylactic acid (PLA), multiwalled carbon nanotubes (MWCNTs), and gelatin nanofibrils (GNFs) coated with the recombinant human erythropoietin-loaded chitosan nanoparticles (rhEpo-CNPs). The PLA/MWCNTs/GNFs/rhEpo-CNPs conduit had the porosity of 85.78 ± 0.70%, the contact angle of 77.65 ± 1.91° and the ultimate tensile strength and compressive modulus of 5.51 ± 0.13 MPa and 2.66 ± 0.34 MPa, respectively. The conduit showed the electrical conductivity of 0.32 S cm -1 and lost about 11% of its weight after 60 days in normal saline. The produced conduit was able to release the rhEpo for at least 2 weeks and exhibited favorable cytocompatibility towards SCs. For functional analysis, the conduit was seeded with 1.5 × 10 4 SCs and implanted into a 10 mm sciatic nerve defect of Wistar rat. After 14 weeks, the results of sciatic functional index, hot plate latency, compound muscle action potential amplitude, weight-loss percentage of wet gastrocnemius muscle and Histopathological examination using hematoxylin-eosin and Luxol fast blue staining demonstrated that the produced conduit had comparable nerve regeneration to the autograft, as the gold standard to bridge the nerve gaps. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1463-1476, 2018. © 2017 Wiley Periodicals, Inc.

  16. Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

    International Nuclear Information System (INIS)

    Mantovani, Cristina; Raimondo, Stefania; Haneef, Maryam S.; Geuna, Stefano; Terenghi, Giorgio; Shawcross, Susan G.; Wiberg, Mikael

    2012-01-01

    Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker ® staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration. -- Highlights: ► Aged MSC and ASC differentiated into Schwann-like cells support axon regeneration. ► p53 expression does not appreciably influence the biology of Schwann or stem cells. ► Notch 2 expression was similar in cells derived from animals of different ages. ► Proliferation rates of dMSC varied little over time or with animal age.

  17. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

    Science.gov (United States)

    Fitzgerald, Kerry D; Semler, Bert L

    2013-09-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2013-01-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

  19. Origin of microbial life hypothesis: a gel cytoplasm lacking a bilayer membrane, with infrared radiation producing exclusion zone (EZ) water, hydrogen as an energy source and thermosynthesis for bioenergetics.

    Science.gov (United States)

    Trevors, J T; Pollack, G H

    2012-01-01

    The hypothesis is proposed that pre-biotic bacterial cell(s) and the first cells capable of growth/division did not require a cytoplasmic membrane. A gel-like microscopic structure less than a cubic micrometer may have had a dual role as both an ancient pre-cytoplasm and a boundary layer to the higher-entropy external environment. The gel pre-cytoplasm exposed to radiant energy, especially in the infrared (IR) region of the EM spectrum resulted in the production of an exclusion zone (EZ) with a charge differential (-100 to -200 mV) and boundary that may have been a possible location for the latter organization of the first cytoplasmic membrane. Pre-biotic cells and then-living cells may have used hydrogen as the universal energy source, and thermosynthesis in their bioenergetic processes. These components will be discussed as to how they are interconnected, and their hypothesized roles in the origin of life. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  20. Investigation of radiation enhanced reactivation of cytoplasmic replicating human virus

    International Nuclear Information System (INIS)

    Bockstahler, L.E.; Haynes, K.F.; Stafford, J.E.

    1976-01-01

    When monolayers of CV-1 monkey kidney cells were exposed to ultraviolet (uv) radiation (0 to 200 erg/nm 2 ) or x rays (0 to 10 krads) before infection with uv-irradiated herpes simplex virus, an increase in the infectivity of this nuclear replicating virus occurred as measured by plaque formation. These phenomena are known as uv (Weigle) reactivation (WR) and x-ray reactivation (x-ray R). In this study the presence of WR and x-ray R was examined in CV-1 cells infected with uv-irradiated vaccinia virus or poliovirus, both cytoplasmic replicating viruses. Little or no WR or x-ray R was observed for either of these viruses. These results suggest that WR and x-ray R in mammalian cells may be restricted to viruses which are synthesized in the cell nucleus

  1. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

    International Nuclear Information System (INIS)

    Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

    1984-01-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

  2. Promoting peripheral myelin repair

    OpenAIRE

    Zhou, Ye; Notterpek, Lucia

    2016-01-01

    Compared to the central nervous system (CNS), peripheral nerves have a remarkable ability to regenerate and remyelinate. This regenerative capacity to a large extent is dependent on and supported by Schwann cells, the myelin-forming glial cells of the peripheral nervous system (PNS). In a variety of paradigms, Schwann cells are critical in the removal of the degenerated tissue, which is followed by remyelination of newly-regenerated axons. This unique plasticity of Schwann cells has been the ...

  3. Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto.

    Science.gov (United States)

    Pan, Hung-Chuan; Yang, Dar-Yu; Ho, Shu-Peng; Sheu, Meei-Ling; Chen, Chung-Jung; Hwang, Shiaw-Min; Chang, Ming-Hong; Cheng, Fu-Chou

    2009-08-23

    Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

  4. Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto

    Directory of Open Access Journals (Sweden)

    Pan Hung-Chuan

    2009-08-01

    Full Text Available Abstract Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days; Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

  5. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    International Nuclear Information System (INIS)

    Shimura, Takaya; Higashiyama, Shigeki; Joh, Takashi; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi

    2012-01-01

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for

  6. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Science.gov (United States)

    2012-01-01

    Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Conclusions Both the function of HB-EGF as an EGFR ligand

  7. The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments.

    Science.gov (United States)

    Obruca, Stanislav; Sedlacek, Petr; Mravec, Filip; Krzyzanek, Vladislav; Nebesarova, Jana; Samek, Ota; Kucera, Dan; Benesova, Pavla; Hrubanova, Kamila; Milerova, Miluse; Marova, Ivana

    2017-10-25

    Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

    Science.gov (United States)

    Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

    2015-02-18

    Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. pH-Responsive Micelle-Based Cytoplasmic Delivery System for Induction of Cellular Immunity

    Directory of Open Access Journals (Sweden)

    Eiji Yuba

    2017-11-01

    Full Text Available (1 Background: Cytoplasmic delivery of antigens is crucial for the induction of cellular immunity, which is an important immune response for the treatment of cancer and infectious diseases. To date, fusogenic protein-incorporated liposomes and pH-responsive polymer-modified liposomes have been used to achieve cytoplasmic delivery of antigen via membrane rupture or fusion with endosomes. However, a more versatile cytoplasmic delivery system is desired for practical use. For this study, we developed pH-responsive micelles composed of dilauroyl phosphatidylcholine (DLPC and deoxycholic acid and investigated their cytoplasmic delivery performance and immunity-inducing capability. (2 Methods: Interaction of micelles with fluorescence dye-loaded liposomes, intracellular distribution of micelles, and antigenic proteins were observed. Finally, antigen-specific cellular immune response was evaluated in vivo using ELIspot assay. (3 Results: Micelles induced leakage of contents from liposomes via lipid mixing at low pH. Micelles were taken up by dendritic cells mainly via macropinocytosis and delivered ovalbumin (OVA into the cytosol. After intradermal injection of micelles and OVA, OVA-specific cellular immunity was induced in the spleen. (4 Conclusions: pH-responsive micelles composed of DLPC and deoxycholic acid are promising as enhancers of cytosol delivery of antigens and the induction capability of cellular immunity for the treatment of cancer immunotherapy and infectious diseases.

  10. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

    International Nuclear Information System (INIS)

    Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

    2008-01-01

    The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

  11. The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells

    Directory of Open Access Journals (Sweden)

    Zeyou Wang

    2016-11-01

    Full Text Available Abstract Background As a well-characterized key player in various signal transduction networks, extracellular-signal-regulated kinase (ERK1/2 has been widely implicated in the development of many malignancies. We previously found that Leucine-rich repeat containing 4 (LRRC4 was a tumor suppressor and a negative regulator of the ERK/MAPK pathway in glioma tumorigenesis. However, the precise molecular role of LRRC4 in ERK signal transmission is unclear. Methods The interaction between LRRC4 and ERK1/2 was assessed by co-immunoprecipitation and GST pull-down assays in vivo and in vitro. We also investigated the interaction of LRRC4 and ERK1/2 and the role of the D domain in ERK activation in glioma cells. Results Here, we showed that LRRC4 and ERK1/2 interact via the D domain and CD domain, respectively. Following EGF stimuli, the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and abrogates ERK1/2 activation and nuclear translocation. In glioblastoma cells, ectopic LRRC4 expression competitively inhibited the interaction of endogenous mitogen-activated protein kinase (MEK and ERK1/2. Mutation of the D domain decreased the LRRC4-mediated inhibition of MAPK signaling and its anti-proliferation and anti-invasion roles. Conclusions Our results demonstrated that the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells. These findings identify a new mechanism underlying glioblastoma progression and suggest a novel therapeutic strategy by restoring the activity of LRRC4 to decrease MAPK cascade activation.

  12. Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB

    Science.gov (United States)

    Soh, Hendrick; Venkatesan, Natarajan; Veena, Mysore S.; Ravichandran, Sandhiya; Zinabadi, Alborz; Basak, Saroj K.; Parvatiyar, Kislay; Srivastava, Meera; Liang, Li-Jung; Gjertson, David W.; Torres, Jorge Z.; Moatamed, Neda A.

    2016-01-01

    We and others have shown that the cystatin E/M gene is inactivated in primary human tumors, pointing to its role as a tumor suppressor gene. However, the molecular mechanism of tumor suppression is not yet understood. Using plasmid-directed cystatin E/M gene overexpression, a lentivirus-mediated tetracycline-inducible vector system, and human papillomavirus 16 (HPV 16) E6 and E7 gene-immortalized normal human epidermal keratinocytes, we demonstrated intracellular and non-cell-autonomous apoptotic growth inhibition of tumor cell lines and that growth inhibition is associated with cytoplasmic retention of NF-κB. We further demonstrated decreased phosphorylation of IκB kinase (IKKβ) and IκBα in the presence of tumor necrosis factor alpha (TNF-α), confirming the role of cystatin E/M in the regulation of the NF-κB signaling pathway. Growth suppression of nude mouse xenograft tumors carrying a tetracycline-inducible vector system was observed with the addition of doxycycline in drinking water, confirming that the cystatin E/M gene is a tumor suppressor gene. Finally, immunohistochemical analyses of cervical carcinoma in situ and primary tumors have shown a statistically significant inverse relationship between the expression of cystatin E/M and cathepsin L and a direct relationship between the loss of cystatin E/M expression and nuclear expression of NF-κB. We therefore propose that the cystatin E/M suppressor gene plays an important role in the regulation of NF-κB. PMID:27090639

  13. Cytoplasmic CXCR4 expression in breast cancer: induction by nitric oxide and correlation with lymph node metastasis and poor prognosis

    International Nuclear Information System (INIS)

    Yasuoka, Hironao; Tsujimoto, Masahiko; Yoshidome, Katsuhide; Nakahara, Masaaki; Kodama, Rieko; Sanke, Tokio; Nakamura, Yasushi

    2008-01-01

    Lymph nodes constitute the first site of metastasis for most malignancies, and the extent of lymph node involvement is a major criterion for evaluating patient prognosis. The CXC chemokine receptor 4 (CXCR4) has been shown to play an important role in lymph node metastasis. Nitric oxide (NO) may also contribute to induction of metastatic ability in human cancers. CXCR4 expression was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels (a biomarker for peroxynitrate formation from NO in vivo) and lymph node status, CXCR4 immunoreactivity, and other established clinico-pathological parameters, as well as prognosis, was analyzed. Nitrite/nitrate levels and CXCR4 expressions were assessed in MDA-MB-231 and SK-BR-3 breast cancer cell lines after induction and/or inhibition of NO synthesis. CXCR4 staining was predominantly cytoplasmic; this was observed in 50%(56/113) of the tumors. Cytoplasmic CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis. Kaplan-Meier survival curves showed that cytoplasmic CXCR4 expression was associated with reduced disease-free and overall survival. In multivariate analysis, cytoplasmic CXCR4 expression emerged as a significant independent predictor for overall and disease-free survival. Cytoplasmic expression of functional CXCR4 in MDA-MB-231 and SK-BR-3 cells was increased by treatment with the NO donor DETA NONOate. This increase was abolished by L-NAME, an inhibitor of NOS. Our data showed a role for NO in stimulating cytoplasmic CXCR4 expression in vitro. Formation of the biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in vivo. In addition, cytoplasmic CXCR4 expression may serve as a significant prognostic factor for long-term survival in breast cancer

  14. Evidence for significantly enhancing reduction of Azo dyes in Escherichia coli by expressed cytoplasmic Azoreductase (AzoA) of Enterococcus faecalis.

    Science.gov (United States)

    Feng, J; Heinze, T M; Xu, H; Cerniglia, C E; Chen, H

    2010-05-01

    Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

  15. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP).

    Science.gov (United States)

    Baratella, Marco; Forlani, Greta; Raval, Goutham U; Tedeschi, Alessandra; Gout, Olivier; Gessain, Antoine; Tosi, Giovanna; Accolla, Roberto S

    2017-01-01

    HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

  16. Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP.

    Directory of Open Access Journals (Sweden)

    Marco Baratella

    2017-01-01

    Full Text Available HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL, and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP. Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

  17. Cytoplasmic Drosha Is Aberrant in Precancerous Lesions of Gastric Carcinoma and Its Loss Predicts Worse Outcome for Gastric Cancer Patients.

    Science.gov (United States)

    Zhang, Hailong; Hou, Yixuan; Xu, Liyun; Zeng, Zongyue; Wen, Siyang; Du, Yan-E; Sun, Kexin; Yin, Jiali; Lang, Lei; Tang, Xiaoli; Liu, Manran

    2016-04-01

    The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume (P = 0.002), tumor grade (P gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.

  18. Assessment of the Nucleus-to-Cytoplasmic Ratio in MCF-7 Cells Using Ultra-high Frequency Ultrasound and Photoacoustics

    Science.gov (United States)

    Moore, M. J.; Strohm, E. M.; Kolios, M. C.

    2016-12-01

    The nucleus-to-cytoplasmic (N:C) ratio of a cell is often used when assessing histology for the presence of malignant disease. In this proof of concept study, we present a new, non-optical method for determination of the N:C ratio using ultra-high Frequency ultrasound (US) and photoacoustics (PA). When using transducers in the 100 MHz-500 MHz range, backscattered US pulses and emitted PA waves are encoded with information pertaining to the dimension and morphology of micron-sized objects. If biological cells are interrogated, the diameter of the scattering or absorbing structure can be assessed by fitting the power spectra of the measured US or PA signals to theoretical models for US backscatter and PA emission from a fluid sphere. In this study, the cell and nucleus diameters of 9 MCF-7 breast cancer cells were determined using a new simplified model that calculates the theoretical values of the location of the power spectra minima for both US and PA signals. These diameters were then used to calculate the N:C ratio of the measured cells. The average cell diameter determined by US pulses from a transducer with a central frequency of 375 MHz was found to be 15.5 μ m± 1.8 μ m. The PA waves emitted by the cell nuclei were used to determine an average nuclear diameter of 12.0 μ m± 1.3 μ m. The N:C ratio for these cells was calculated to be 1.9± 1.0, which agrees well with previously reported N:C values for this cell type.

  19. Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

    Energy Technology Data Exchange (ETDEWEB)

    Mantovani, Cristina [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Department of Integrative Medical Biology and Surgical and Perioperative Science, Umea University, Umea (Sweden); Department of Surgical and Perioperative Science, Umea University, Umea (Sweden); Raimondo, Stefania [Dipartimento di Scienze Cliniche e Biologiche, University of Turin (Italy); Haneef, Maryam S. [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Geuna, Stefano [Dipartimento di Scienze Cliniche e Biologiche, University of Turin (Italy); Terenghi, Giorgio [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Shawcross, Susan G., E-mail: sue.shawcross@manchester.ac.uk [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Wiberg, Mikael [Department of Integrative Medical Biology and Surgical and Perioperative Science, Umea University, Umea (Sweden); Department of Surgical and Perioperative Science, Umea University, Umea (Sweden)

    2012-10-01

    Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker{sup Registered-Sign} staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration. -- Highlights: Black-Right-Pointing-Pointer Aged MSC and ASC differentiated into Schwann-like cells support axon regeneration. Black-Right-Pointing-Pointer p53 expression does not appreciably influence the biology of Schwann or stem cells. Black-Right-Pointing-Pointer Notch 2 expression was similar in cells derived from animals of different ages. Black-Right-Pointing-Pointer Proliferation rates of dMSC varied little over time or with animal age.

  20. Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane

    NARCIS (Netherlands)

    Wenzel, M.; Kohl, B.; Münch, D.; Raatschen, N.; Albada, H.B.; Hamoen, L.; Metzler-Nolte, N.; Sahl, H.G.; Bandow, J.E.

    2012-01-01

    Mersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end

  1. Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

    Science.gov (United States)

    Rincheval, Vincent; Lelek, Mickael; Gault, Elyanne; Bouillier, Camille; Sitterlin, Delphine; Blouquit-Laye, Sabine; Galloux, Marie; Zimmer, Christophe; Eleouet, Jean-François; Rameix-Welti, Marie-Anne

    2017-09-15

    Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

  2. Direct Cytoplasmic Delivery and Nuclear Targeting Delivery of HPMA-MT Conjugates in a Microtubules Dependent Fashion.

    Science.gov (United States)

    Zhong, Jiaju; Zhu, Xi; Luo, Kui; Li, Lian; Tang, Manlin; Liu, Yanxi; Zhou, Zhou; Huang, Yuan

    2016-09-06

    As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve direct cytoplasmic delivery by a nonendocytic pathway and transport into the nucleus in a microtubules dependent fashion. A special targeting peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into the nucleus a by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by a nonendocytic pathway, thus avoiding the drug degradation in the lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the DTX and Noc treated groups. These results demonstrated that the conjugates transported into the nucleus in a microtubules dependent way. Therefore, in addition to direct cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

  3. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    International Nuclear Information System (INIS)

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-01-01

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions

  4. Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing.

    Science.gov (United States)

    Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

    2015-01-01

    Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection.

  5. Rapid and Sustained Nuclear-Cytoplasmic ERK Oscillations Induced by Epidermal Growth Factor

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Harish; Ippolito, Danielle L.; Chrisler, William B.; Resat, Haluk; Bollinger, Nikki; Opresko, Lee K.; Wiley, H. S.

    2009-12-01

    Mathematical modeling has predicted that ERK activity should oscillate in response to cell stimulation, but this has never been observed. To explore this inconsistency, we expressed an ERK1-GFP fusion protein in mammary epithelial cells. Following EGF stimulation, we observed rapid and continuous ERK oscillations between the nucleus and cytoplasm with a periodicity of approximately 15 minutes. These oscillations were remarkably persistent (>45 cycles), displayed an asymmetric waveform, and were highly dependent on cell density, essentially disappearing at confluency. We conclude that the ERK pathway is an intrinsic oscillator. Although the functional implications of the observed oscillations are uncertain, this property can be used to continuously monitor ERK activity in single cells.

  6. Effects of cavernous nerve reconstruction on expression of nitric oxide synthase isoforms in rats.

    Science.gov (United States)

    Schlenker, Boris; Matiasek, Kaspar; Saur, Dieter; Gratzke, Christian; Bauer, Ricarda M; Herouy, Yared; Arndt, Christian; Blesch, Armin; Hartung, Rudolf; Stief, Christian G; Weidner, Norbert; May, Florian

    2010-12-01

    To evaluate the expression of nitric oxide synthase (NOS) isoforms after various reconstruction techniques in rats, to improve the understanding of neuronal repair mechanisms after radical prostatectomy, as Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and glial cell-line-derived neurotrophic factor (GDNF)-overexpressing Schwann cells enhance nerve regenerative capacity. Segments (5 mm) of the cavernous nerve were excised bilaterally, followed by immediate bilateral microsurgical reconstruction. In four rats per group, the eight nerves were reconstructed by autologous nerve grafting (A), interposition of Schwann cell-seeded silicon tubes (B), or silicon tubes seeded with GDNF-hypersecreting Schwann cells (C). Further rats were either sham-operated (D) or had nerve excision without repair (E). Erectile function was evaluated after 6 weeks by re-laparotomy, electrical nerve stimulation and morphological evaluation of reconstructed nerves. NOS isoform mRNA expression was analysed by reverse transcription-polymerase chain reaction in tissue specimens taken from the corpora cavernosa. GDNF-transduced Schwann cell grafts restored erectile function better than untransduced Schwann cell and autologous nerve grafts (88% vs 75% vs 38%; not significant). Tissue specimens in group C had the highest expression of neuronal NOS mRNA in relation to the neuronal marker PGP9.5 among all treatment groups (not significant). Compared to nerve grafts (A) and negative controls (E) nNOS/PGP9.5 expression was significantly higher (P Schwann cell grafts (P < 0.05). Restoration of erectile function is paralleled by an increase of neuronal NOS expression in rats. Further experiments will determine the physiological role of neuronal NOS in erectile nerve repair processes. © 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.

  7. [Role of erythrocyte cytoplasmic structures in changes in the affinity of haemoglobin for oxygen].

    Science.gov (United States)

    Bryzgalova, N Iu; Brazhe, N A; Iusipovich, A U; Maksimov, G V; Rubin, A B

    2009-01-01

    Changes in the refractive index of the cytoplasm and the affinity of haemoporphyrin of erythrocyte haemoglobin to oxygen (pH, 2,3-diphosphoglycerate) have been investigated using laser interference microscopy and Raman spectroscopy. It has been established that a decrease in pH and an increase in the content of 2,3-diphosphoglycerate are accompanied by changes in both the form of the cell and the refractive index of the cytoplasm and the affinity of haemoporphyrin of hemoglobin to oxygen. It has been shown that as pH is reduced, the capacity of haemoporphyrin for binding oxygen decreases and as the concentration of 2,3-diphosphoglycerate is increased, the ability of haemoporphyrin for oxygen reabsorption increases.

  8. The effect of the external medium on the gravity-induced polarity of cytoplasmic streaming in Chara corallina (Characeae)

    Science.gov (United States)

    Staves, M. P.; Wayne, R.; Leopold, A. C.

    1997-01-01

    Gravity induces a polarity of cytoplasmic streaming in vertical internodal cells of Chara such that the downwardly directed stream moves faster than the upwardly directed stream. In order to determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best explain the gravity response in Chara internodal cells, we controlled the physical properties of the external medium, including density and osmolarity, with impermeant solutes and examined the effect on the polarity of cytoplasmic streaming. As the density of the external medium is increased, the polarity of cytoplasmic streaming decreases and finally disappears when the density of the external medium is equal to that of the cell (1015 kg/m3). A further increase in the density of the external medium causes a reversal of the gravity response. These results are consistent with the gravitational pressure theory of gravity sensing since the buoyancy of the protoplast is dependent on the difference between the density of the protoplast and the external medium, and are inconsistent with the statolith theory since the buoyancy of intracellular particles are unaffected by changes in the external medium.

  9. Prediction of Chlamydia pneumoniae protein localization in host mitochondria and cytoplasm and possible involvements in lung cancer etiology: a computational approach

    Directory of Open Access Journals (Sweden)

    Aws Alshamsan

    2017-12-01

    Full Text Available Collecting evidence suggests that the intercellular infection of Chlamydia pneumoniae in lungs contributes to the etiology of lung cancer. Many proteins of Chlamydia pneumoniae outmanoeuvre the various system of the host. The infection may regulate various factors, which can influence the growth of lung cancer in affected persons. In this in-silico study, we predict potential targeting of Chlamydia pneumoniae proteins in mitochondrial and cytoplasmic comportments of host cell and their possible involvement in growth and development of lung cancer. Various cellular activities are controlled in mitochondria and cytoplasm, where the localization of Chlamydia pneumoniae proteins may alter the normal functioning of host cells. The rationale of this study is to find out and explain the connection between Chlamydia pneumoniae infection and lung cancer. A sum of 183 and 513 proteins were predicted to target in mitochondria and cytoplasm of host cell out of total 1112 proteins of Chlamydia pneumoniae. In particular, many targeted proteins may interfere with normal growth behaviour of host cells, thereby altering the decision of program cell death. Present article provides a potential connection of Chlamydia pneumoniae protein targeting and proposed that various targeted proteins may play crucial role in lung cancer etiology through diverse mechanisms.

  10. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Directory of Open Access Journals (Sweden)

    Shimura Takaya

    2012-05-01

    Full Text Available Abstract Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C, translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF and mutated HB-EGF (HB-EGF-mC, which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 % and in the cytoplasm only in 25 cases (26.0 %. The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P  Conclusions Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation might be crucial in gastric cancer invasion. HB-EGF-C nuclear translocation may offer a prognostic marker and a new molecular target for gastric cancer therapy.

  11. The intermediate filament protein vimentin binds specifically to a recombinant integrin α2/β1 cytoplasmic tail complex and co-localizes with native α2/β1 in endothelial cell focal adhesions

    International Nuclear Information System (INIS)

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly

    2005-01-01

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen

  12. Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

    Directory of Open Access Journals (Sweden)

    Antonio Postigo

    2017-05-01

    Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

  13. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    Directory of Open Access Journals (Sweden)

    Mayumi F. Kohiyama

    2015-01-01

    Full Text Available Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by a coding polyglutamine expansion in the Ataxin-1 gene ( ATXN1 , which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

  14. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

    International Nuclear Information System (INIS)

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2015-01-01

    is predominantly present in the cytoplasm and the survival of the Jak2(V617F) + HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5

  15. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1–CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1. PMID:25694549

  16. Comparison of Gene Expression Profiles in Human Germinal Vesicle Before and After Cytoplasmic Transfer From Mature Oocytes in Iranian Infertile Couples

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    Fatemeh Sadat Hoseini

    2016-08-01

    Full Text Available Objective: To evaluate the effect of cytoplasm transfer from mature oocytes to germinal vesicle(GVs on promoting the maturation of cytoplasm of GV at the mRNA level.Materials and methods: Sixty six in vitro fertilization (IVF operations between June 2012 and November 2013 were included in this study. Totally 120 GVs were obtained. Normal GVs were categorized into 3 groups (n = 40 randomly: the first comprised oocytes that did not receive the cytoplasm of mature oocytes; the second group comprised oocytes that did not receive the cytoplasm of mature oocytes but were incubated for 24 h; and the third group comprised oocytes that received 10-15% the cytoplasm of mature oocytes and were then incubated for 24 h. Each group was separately analyzed by quantitative polymerase chain reaction (qPCR and the expression levels of selected genes were assessed.Results: The expression levels of genes involved in the cytoplasmic maturity, and energy-producing mitochondria were significantly higher in the pooled oocytes of 2nd control group than those of the 1st control and intervention groups (p < 0.001. The genes involved in the meiosis, spindle check point, DNA repairing and cell cycle checkpoint did not have any expression in the 1st and intervention groups; however, these genes were expressed in the 2nd group, significantly. In the 2nd group, the highest expression level was observed for genes involved in the DNA repairing and cell cycle checkpoint. In the intervention group, none of the genes were expressed except for energy-producing mitochondria gene; even in this case, the expression level of this gene in this group of oocytes was significantly lower than that in other groups (p < 0.001. After 24 h meiosis assumption was significantly higher in the third group than in the second group (95% vs. 68%, p < 0.001.Conclusion: The cytoplasm transfer technique is not effective in cytoplasmic maturity of the recipient GV oocytes. In contrast, 24-hr in

  17. Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Kong

    Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

  18. Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

    Science.gov (United States)

    Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke; Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho; Yamamoto, Akitsugu; Nakamura, Nobuhiro

    2014-11-01

    The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  20. In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering

    OpenAIRE

    Wrobel, Sandra; Serra, Sofia Cristina; Samy, S. M.; Sousa, Nuno; Heimann, Claudia; Barwig, Christina; Grothe, Claudia; Salgado, A. J.; Talini, Kirsten Haastert

    2014-01-01

    Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)-immortalized, neonatal, and adult-as well as rat bone-marrow-derived mesenchymal stromal cells (BMSC...

  1. The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes

    International Nuclear Information System (INIS)

    Greer, Amy E.; Hearing, Patrick; Ketner, Gary

    2011-01-01

    The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 k in viral late mRNA accumulation.

  2. First cytoplasmic loop of glucagon-like peptide-1 receptor can function at the third cytoplasmic loop position of rhodopsin.

    Science.gov (United States)

    Yamashita, Takahiro; Tose, Koji; Shichida, Yoshinori

    2008-01-01

    G protein-coupled receptors (GPCRs) are classified into several families based on their amino acid sequences. In family 1, GPCRs such as rhodopsin and adrenergic receptor, the structure-function relationship has been extensively investigated to demonstrate that exposure of the third cytoplasmic loop is essential for selective G protein activation. In contrast, much less is known about other families. Here we prepared chimeric mutants between Gt-coupled rhodopsin and Gi/Go- and Gs-coupled glucagon-like peptide-1 (GLP-1) receptor of family 2 and tried to identify the loop region that functions at the third cytoplasmic loop position of rhodopsin. We succeeded in expressing a mutant having the first cytoplasmic loop of GLP-1 receptor and found that this mutant activated Gi and Go efficiently but did not activate Gt. Moreover, the rhodopsin mutant having the first loop of Gs-coupled secretin receptor of family 2 decreased the Gi and Go activation efficiencies. Therefore, the first loop of GLP-1 receptor would share a similar role to the third loop of rhodopsin in G protein activation. This result strongly suggested that different families of GPCRs have maintained molecular architectures of their ancestral types to generate a common mechanism, namely exposure of the cytoplasmic loop, to activate peripheral G protein.

  3. A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

    Science.gov (United States)

    Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

    2016-05-10

    The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

  4. The role of undifferentiated adipose-derived stem cells in peripheral nerve repair.

    Science.gov (United States)

    Zhang, Rui; Rosen, Joseph M

    2018-05-01

    Peripheral nerve injuries impose significant health and economic consequences, yet no surgical repair can deliver a complete recovery of sensory or motor function. Traditional methods of repair are less than ideal: direct coaptation can only be performed when tension-free repair is possible, and transplantation of nerve autograft can cause donor-site morbidity and neuroma formation. Cell-based therapy delivered via nerve conduits has thus been explored as an alternative method of nerve repair in recent years. Stem cells are promising sources of the regenerative core material in a nerve conduit because stem cells are multipotent in function, abundant in supply, and more accessible than the myelinating Schwann cells. Among different types of stem cells, undifferentiated adipose-derived stem cell (uASC), which can be processed from adipose tissue in less than two hours, is a promising yet underexplored cell type. Studies of uASC have emerged in the past decade and have shown that autologous uASCs are non-immunogenic, easy to access, abundant in supply, and efficacious at promoting nerve regeneration. Two theories have been proposed as the primary regenerative mechanisms of uASC: in situ trans-differentiation towards Schwann cells, and secretion of trophic and anti-inflammatory factors. Future studies need to fully elucidate the mechanisms, side effects, and efficacy of uASC-based nerve regeneration so that uASCs can be utilized in clinical settings.

  5. Promoting peripheral myelin repair.

    Science.gov (United States)

    Zhou, Ye; Notterpek, Lucia

    2016-09-01

    Compared to the central nervous system (CNS), peripheral nerves have a remarkable ability to regenerate and remyelinate. This regenerative capacity to a large extent is dependent on and supported by Schwann cells, the myelin-forming glial cells of the peripheral nervous system (PNS). In a variety of paradigms, Schwann cells are critical in the removal of the degenerated tissue, which is followed by remyelination of newly-regenerated axons. This unique plasticity of Schwann cells has been the target of myelin repair strategies in acute injuries and chronic diseases, such as hereditary demyelinating neuropathies. In one approach, the endogenous regenerative capacity of Schwann cells is enhanced through interventions such as exercise, electrical stimulation or pharmacological means. Alternatively, Schwann cells derived from healthy nerves, or engineered from different tissue sources have been transplanted into the PNS to support remyelination. These transplant approaches can then be further enhanced by exercise and/or electrical stimulation, as well as by the inclusion of biomaterial engineered to support glial cell viability and neurite extension. Advances in our basic understanding of peripheral nerve biology, as well as biomaterial engineering, will further improve the functional repair of myelinated peripheral nerves. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect.

    Science.gov (United States)

    Luo, Lihua; Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan; Wang, Xiong; Huselstein, Celine; Chen, Yun

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  7. Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

    International Nuclear Information System (INIS)

    Poznanski, Jaroslaw; Szczesny, Pawel; Ruszczyńska, Katarzyna; Zielenkiewicz, Piotr; Paczek, Leszek

    2013-01-01

    Highlights: ► We predicted buffering capacity of yeast proteome from protein abundance data. ► We measured total buffering capacity of yeast cytoplasm. ► We showed that proteins contribute insignificantly to buffering capacity. -- Abstract: Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell’s intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

  8. F4/80 inhibits osteoclast differentiation via downregulation of nuclear factor of activated T cells, cytoplasmic 1.

    Science.gov (United States)

    Kang, Ju-Hee; Sim, Jung-Sun; Zheng, Ting; Yim, Mijung

    2017-04-01

    Osteoclastogenesis is an essential process in bone metabolism, which can be induced by RANKL stimulation. The F4/80 glycoprotein is a member of the EGF-transmembrane 7 (TM7) family and has been established as a specific cell-surface marker for murine macrophages. This study aimed to identify the role of F4/80 in osteoclastogenesis. Using mouse bone marrow-derived macrophages (BMMs), we observed that the mRNA level of F4/80 was dramatically reduced as these cells differentiated into osteoclasts. Furthermore, osteoclastogenesis was decreased in F4/80 high BMMs compared to F4/80 -/low BMMs. The inhibitory effect of F4/80 was associated with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Ectopic overexpression of a constitutively active form of NFATc1 rescued the anti-osteoclastogenic effect of F4/80 completely, suggesting that the anti-osteoclastogenic effect of F4/80 was mainly due to reduction in NFATc1 expression. As an underlying mechanism, we demonstrated that the presence of F4/80 abrogated the effect of RANKL on the phosphorylation of CREB and activated the expression of IFN-β, which are restored by cyclic AMP. Collectively, our results demonstrate that the presence of F4/80 suppresses RANKL-induced osteoclastogenesis by impairing the expression of NFATc1 via CREB and IFN-β. Therefore, F4/80 may hold therapeutic potential for bone destructive diseases.

  9. Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

    Science.gov (United States)

    Nicolas, William J; Grison, Magali S; Trépout, Sylvain; Gaston, Amélia; Fouché, Mathieu; Cordelières, Fabrice P; Oparka, Karl; Tilsner, Jens; Brocard, Lysiane; Bayer, Emmanuelle M

    2017-06-12

    Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

  10. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  11. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    International Nuclear Information System (INIS)

    Chowdhury, E.H.

    2011-01-01

    Highlights: → Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. → Fluoridated carbonate apatite promotes a robust increase in transgene expression. → Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  12. Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments.

    Science.gov (United States)

    Kumar, Amit; Bhowmick, Prolay Kumar; Singh, Vikram Jeet; Malik, Manoj; Gupta, Ashish Kumar; Seth, R; Nagarajan, M; Krishnan, S Gopala; Singh, Ashok Kumar

    2017-10-01

    Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4 . Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F 1 s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F 1 s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363

  13. Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

    Science.gov (United States)

    Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

    1999-01-01

    β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

  14. A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

    Science.gov (United States)

    Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

    2014-01-02

    Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.

  15. Cytoplasmic localization of Hug1p, a negative regulator of the MEC1 pathway, coincides with the compartmentalization of Rnr2p–Rnr4p

    Energy Technology Data Exchange (ETDEWEB)

    Ainsworth, William B. [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Hughes, Bridget Todd; Au, Wei Chun; Sakelaris, Sally [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Kerscher, Oliver [Biology Department, The College of William and Mary, Williamsburg, VA 23185 (United States); Benton, Michael G., E-mail: benton@lsu.edu [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Basrai, Munira A., E-mail: basraim@mail.nih.gov [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-10-04

    Highlights: •Hug1p overexpression sensitizes wild-type cells to DNA damage and hydroxyurea (HU). •Expression of Hug1p in response to HU treatment is delayed relative to Rnr3p. •MEC1 pathway genes are required for cytoplasmic localization of Hug1p. •Hug1p subcellular compartmentalization to the cytoplasm coincides with Rnr2p–Rnr4p. -- Abstract: The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p–Rnr4p subunits and inactivation of negative regulators Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p–Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p–Rnr4p.

  16. Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Toshiro [Hokkaido Univ., Sapporo (Japan). Faculty of Agriculture

    1982-03-01

    Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M/sub 4/ generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets.

  17. Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

    International Nuclear Information System (INIS)

    Kinoshita, Toshiro

    1982-01-01

    Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M 4 generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets. (Kaihara, S.)

  18. Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2009-04-01

    Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

  19. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    , Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F){sup +} HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5.

  20. Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete plant innate immune response.

    Directory of Open Access Journals (Sweden)

    Ana V García

    2010-07-01

    Full Text Available An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1. In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.

  1. A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

    Directory of Open Access Journals (Sweden)

    Davide Danovi

    2010-07-01

    Full Text Available Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF, a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.

  2. Targeted deletion of Sox10 by Wnt1-cre defects neuronal migration and projection in the mouse inner ear.

    Directory of Open Access Journals (Sweden)

    YanYan Mao

    Full Text Available Sensory nerves of the brainstem are mostly composed of placode-derived neurons, neural crest-derived neurons and neural crest-derived Schwann cells. This mixed origin of cells has made it difficult to dissect interdependence for fiber guidance. Inner ear-derived neurons are known to connect to the brain after delayed loss of Schwann cells in ErbB2 mutants. However, the ErbB2 mutant related alterations in the ear and the brain compound interpretation of the data. We present here a new model to evaluate exclusively the effect of Schwann cell loss on inner ear innervation. Conditional deletion of the neural crest specific transcription factor, Sox10, using the rhombic lip/neural crest specific Wnt1-cre driver spares Sox10 expression in the ear. We confirm that neural crest-derived cells provide a stop signal for migrating spiral ganglion neurons. In the absence of Schwann cells, spiral ganglion neurons migrate into the center of the cochlea and even out of the ear toward the brain. Spiral ganglion neuron afferent processes reach the organ of Corti, but many afferent fibers bypass the organ of Corti to enter the lateral wall of the cochlea. In contrast to this peripheral disorganization, the central projection to cochlear nuclei is normal. Compared to ErbB2 mutants, conditional Sox10 mutants have limited cell death in spiral ganglion neurons, indicating that the absence of Schwann cells alone contributes little to the embryonic survival of neurons. These data suggest that neural crest-derived cells are dispensable for all central and some peripheral targeting of inner ear neurons. However, Schwann cells provide a stop signal for migratory spiral ganglion neurons and facilitate proper targeting of the organ of Corti by spiral ganglion afferents.

  3. Targeted Deletion of Sox10 by Wnt1-cre Defects Neuronal Migration and Projection in the Mouse Inner Ear

    Science.gov (United States)

    Mao, YanYan; Reiprich, Simone; Wegner, Michael; Fritzsch, Bernd

    2014-01-01

    Sensory nerves of the brainstem are mostly composed of placode-derived neurons, neural crest-derived neurons and neural crest-derived Schwann cells. This mixed origin of cells has made it difficult to dissect interdependence for fiber guidance. Inner ear-derived neurons are known to connect to the brain after delayed loss of Schwann cells in ErbB2 mutants. However, the ErbB2 mutant related alterations in the ear and the brain compound interpretation of the data. We present here a new model to evaluate exclusively the effect of Schwann cell loss on inner ear innervation. Conditional deletion of the neural crest specific transcription factor, Sox10, using the rhombic lip/neural crest specific Wnt1-cre driver spares Sox10 expression in the ear. We confirm that neural crest-derived cells provide a stop signal for migrating spiral ganglion neurons. In the absence of Schwann cells, spiral ganglion neurons migrate into the center of the cochlea and even out of the ear toward the brain. Spiral ganglion neuron afferent processes reach the organ of Corti, but many afferent fibers bypass the organ of Corti to enter the lateral wall of the cochlea. In contrast to this peripheral disorganization, the central projection to cochlear nuclei is normal. Compared to ErbB2 mutants, conditional Sox10 mutants have limited cell death in spiral ganglion neurons, indicating that the absence of Schwann cells alone contributes little to the embryonic survival of neurons. These data suggest that neural crest-derived cells are dispensable for all central and some peripheral targeting of inner ear neurons. However, Schwann cells provide a stop signal for migratory spiral ganglion neurons and facilitate proper targeting of the organ of Corti by spiral ganglion afferents. PMID:24718611

  4. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    International Nuclear Information System (INIS)

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E.

    2005-01-01

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with β-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins

  5. Pediatric Inflammatory Bowel Disease with Cytoplasmic Staining of Antineutrophil Cytoplasmic Antibodies

    Directory of Open Access Journals (Sweden)

    Omar I. Saadah

    2013-01-01

    Full Text Available Background. It is unusual for the antineutrophil cytoplasmic antibody with cytoplasmic pattern (cANCA to present in patients with inflammatory bowel disease (IBD without vasculitis. The purpose of this study was to describe the occurrence and characteristics of pediatrics IBD with cANCA. Methods. A retrospective review of pediatric IBD associated with cANCA serology in patients from King Abdulaziz University Hospital, Saudi Arabia, between September 2002 and February 2012. Results. Out of 131 patients with IBD screened for cANCAs, cANCA was positive in 7 (5.3% patients of whom 4 had ulcerative colitis and 3 had Crohn's disease. The median age was 8.8 years (2–14.8 years. Six (86% were males. Of the 7 patients, 5 (71% were Saudi Arabians and 2 were of Indian ethnicity. The most common symptoms were diarrhea, abdominal pain, weight loss, and rectal bleeding. None had family history or clinical features suggestive of vasculitis involving renal and respiratory systems. No difference in the disease location or severity was observed between cANCA positive and cANCA negative patients apart from male preponderance in cANCA positive patients. Conclusion. The occurrence of cANCA in pediatric IBD is rare. Apart from male preponderance, there were no peculiar characteristics for the cANCA positive patients.

  6. Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

    Directory of Open Access Journals (Sweden)

    Ashley T Martino

    2009-08-01

    Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

  7. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

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    Cecilia Zampedri

    2016-10-01

    Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

  8. Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves

    OpenAIRE

    Amoh, Yasuyuki; Li, Lingna; Campillo, Raul; Kawahara, Katsumasa; Katsuoka, Kensei; Penman, Sheldon; Hoffman, Robert M.

    2005-01-01

    The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but ne...

  9. Low-energy Shock Wave Therapy Ameliorates Erectile Dysfunction in a Pelvic Neurovascular Injuries Rat Model.

    Science.gov (United States)

    Li, Huixi; Matheu, Melanie P; Sun, Fionna; Wang, Lin; Sanford, Melissa T; Ning, Hongxiu; Banie, Lia; Lee, Yung-Chin; Xin, Zhongcheng; Guo, Yinglu; Lin, Guiting; Lue, Tom F

    2016-01-01

    Erectile dysfunction (ED) caused by pelvic injuries is a common complication of civil and battlefield trauma with multiple neurovascular factors involved, and no effective therapeutic approach is available. To test the effect and mechanisms of low-energy shock wave (LESW) therapy in a rat ED model induced by pelvic neurovascular injuries. Thirty-two male Sprague-Dawley rats injected with 5-ethynyl-2'-deoxyuridine (EdU) at newborn were divided into 4 groups: sham surgery (Sham), pelvic neurovascular injury by bilateral cavernous nerve injury and internal pudendal bundle injury (PVNI), PVNI treated with LESW at low energy (Low), and PVNI treated with LESW at high energy (High). After LESW treatment, rats underwent erectile function measurement and the tissues were harvested for histologic and molecular study. To examine the effect of LESW on Schwann cells, in vitro studies were conducted. The intracavernous pressure (ICP) measurement, histological examination, and Western blot (WB) were conducted. Cell cycle, Schwann cell activation-related markers were examined in in vitro experiments. LESW treatment improves erectile function in a rat model of pelvic neurovascular injury by leading to angiogenesis, tissue restoration, and nerve generation with more endogenous EdU(+) progenitor cells recruited to the damaged area and activation of Schwann cells. LESW facilitates more complete re-innervation of penile tissue with regeneration of neuronal nitric oxide synthase (nNOS)-positive nerves from the MPG to the penis. In vitro experiments demonstrated that LESW has a direct effect on Schwann cell proliferation. Schwann cell activation-related markers including p-Erk1/2 and p75 were upregulated after LESW treatment. LESW-induced endogenous progenitor cell recruitment and Schwann cell activation coincides with angiogenesis, tissue, and nerve generation in a rat model of pelvic neurovascular injuries. Copyright © 2016 International Society for Sexual Medicine. Published by

  10. The RanGTP pathway: from nucleo-cytoplasmic transport to spindle assembly and beyond

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    Tommaso eCavazza

    2016-01-01

    Full Text Available The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context.

  11. APC senses cell-cell contacts and moves to the nucleus upon their disruption.

    Science.gov (United States)

    Brocardo, M G; Bianchini, M; Radrizzani, M; Reyes, G B; Dugour, A V; Taminelli, G L; Gonzalez Solveyra, C; Santa-Coloma, T A

    2001-06-22

    The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored. Copyright 2001 Academic Press.

  12. Clinical Evaluation of Decellularized Nerve Allograft with Autologous Bone Marrow Stem Cells to Improve Peripheral Nerve Repair and Functional Outcomes

    Science.gov (United States)

    2017-07-01

    with autologous mesenchymal stem cells . Exp Neurol. 2007 Apr; 204(2):658-66. 19. Dezawa M., et al., Sciatic nerve regeneration in rats induced by...36 23. Mimura T., et al., Peripheral nerve regeneration by transplantation of bone marrow stromal cell -derived Schwann cells in adult rats. J...AWARD NUMBER: W81XWH-15-2-0026 TITLE: Clinical Evaluation of Decellularized Nerve Allograft with Autologous Bone Marrow Stem Cells to Improve

  13. Anticorpos contra o citoplasma de neutrófilos Antineutrophil cytoplasmic antibodies

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    Ari Stiel Radu

    2005-07-01

    Full Text Available A descoberta do marcador sorológico denominado anticorpo anticitoplasma de neutrófilos revolucionou o diagnóstico e o seguimento das vasculites pulmonares, especialmente da granulomatose de Wegener. Seu padrão pode ser citoplasmático e perinuclear. Sua titulação auxilia no diagnóstico e no seguimento das vasculites pulmonares.The discovery of the serological markers known as antineutrophil cytoplasmic antibodies revolutionized the diagnosis and follow-up treatment of the various forms of pulmonary vasculitis, especially that of Wegener's granulomatosis. The antineutrophil cytoplasmic antibodies pattern can be cytoplasmic or perinuclear. Determination of antineutrophil cytoplasmic antibodies titers aids the diagnosis and follow-up treatment of pulmonary vasculitis.

  14. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

    International Nuclear Information System (INIS)

    Hashimoto, Muneaki; Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou; Inoue, Masayuki; Kita, Kiyoshi; Harada, Shigeharu; Tanaka, Akiko; Aoki, Takashi; Nara, Takeshi

    2012-01-01

    Highlights: ► We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. ► Disruption of the cpsII gene significantly reduced the growth of epimastigotes. ► In particular, the CPSII-null mutant severely retarded intracellular growth. ► The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes—an insect form—possess both activities, amastigotes—an intracellular replicating form of T. cruzi—are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  15. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available Hundreds of double homeobox (DUX genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD. In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain and DUX1 (which is limited to the double homeodomain. Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs. Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs

  16. Ultrastructure of sensory nerve terminals in the penis in green monkey (Cercopithecus aethiops sabaeus).

    Science.gov (United States)

    Malinovský, L

    1977-01-01

    Tje paper describes the ultrastructure of axons in the endings of various types from the corium in the glans penis in green monkey. In the Meissner's endings the axons are mostly completely enveloped in the plasma of Schwann cells. They contain numerous mitochondria which are partially vacuolated or are quite converted into vacuoles. Next, there are pseudomyelinated figures, light vesicles and further organelles. In the papillar simple glomerular endings with accumulation of Schwann cells there are axons irregular in shape, eccentrically placed in the plasma of Schwann cell, rounded smaller axons either completely or partially surrounded by the plasma of Schwann cell and finally axons with a concentric system of lamellae up to four in number, In the complicated glomerular endings the axons vary in appearance and are enveloped in one to five lamellae of Schwann cells, which is typical of those formations. About some of these systems there is a sign of a capsule formed by an elongated lamella probably of the perineurium. When the axons are not enveloped in the plasma of Schwann cell, they are covered by the basement membrane. In close neighbourhood of the epidermis so-called free endings forming groups were found. The plasma of Schwann cell covers them either partially or completely or it again forms around them a lamellar system amounting up to four layers. It is noticeable that these axons are very poor in organelles. A comparison of the simple sensory corpuscles in the nose skin in hedgehog, the funtional properties of Meissner's endings and the simple corpuscles results in the view that the complexes having a larger amount of lamellae correspond to an extent to the simple sensory corpuscles ant that the Meissner's endings and the complicated glomerules are probably a morphological and functional equivalent of simple sensory corpuscles in the non-primate mammals and that the gloverular endings may also be the first (developmental) stage of the simple sensory

  17. Characterization of Novel Cytoplasmic PARP in the Brain of Octopus vulgaris

    Science.gov (United States)

    DE LISA, EMILIA; DE MAIO, ANNA; MOROZ, LEONID L.; MOCCIA, FRANCESCO; MENNELLA, MARIA ROSARIA FARAONE; DI COSMO, ANNA

    2014-01-01

    Recent investigation has focused on the participation of the poly (ADP-ribose) polymerase (PARP) reaction in the invertebrate central nervous system (CNS) during the process of long-term memory (LTM). In this paper, we characterize, localize, and assign a possible role to a cytoplasmic PARP in the brain of Octopus vulgaris. PARP activity was assayed in optic lobes, supraesophageal mass, and optic nerves. The highest levels of enzyme were found in the cytoplasmic fraction. Hyper-activation of the enzyme was detected in Octopus brain after visual discrimination training. Finally, cytoplasmic PARP was found to inhibit Octopus vulgaris actin polymerization. We propose that the cytoplasmic PARP plays a role in vivo to induce the cytoskeletonal reorganization that occurs during learning-induced neuronal plasticity. PMID:22815366

  18. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

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    Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tokyo.ac.jp; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-15

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.

  19. Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

    International Nuclear Information System (INIS)

    Isobe, Ichiro; Maeno, Yoshitaka; Nagao, Masataka; Iwasa, Mineo; Koyama, Hiroyoshi; Seko-Nakamura, Yoshimi; Monma-Ohtaki, Jun

    2003-01-01

    There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

  20. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Enrique Álvarez

    Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.