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Sample records for axonal protein synthesis

  1. Intra-axonal Synthesis of SNAP25 Is Required for the Formation of Presynaptic Terminals

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    Andreia F.R. Batista

    2017-09-01

    Full Text Available Localized protein synthesis is a mechanism for developing axons to react acutely and in a spatially restricted manner to extracellular signals. As such, it is important for many aspects of axonal development, but its role in the formation of presynapses remains poorly understood. We found that the induced assembly of presynaptic terminals required local protein synthesis. Newly synthesized proteins were detectable at nascent presynapses within 15 min of inducing synapse formation in isolated axons. The transcript for the t-SNARE protein SNAP25, which is required for the fusion of synaptic vesicles with the plasma membrane, was recruited to presynaptic sites and locally translated. Inhibition of intra-axonal SNAP25 synthesis affected the clustering of SNAP25 and other presynaptic proteins and interfered with the release of synaptic vesicles from presynaptic sites. This study reveals a critical role for the axonal synthesis of SNAP25 in the assembly of presynaptic terminals.

  2. Golgi bypass for local delivery of axonal proteins, fact or fiction?

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    González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

    2018-04-06

    Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

  3. In vivo phosphorylation of axonal proteins in goldfish optic nerve during regeneration

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    Larrivee, D.C.; Grafstein, B.

    1987-01-01

    In vivo phosphorylation of axonal proteins was investigated in normal and regenerating optic nerves of goldfish by two-dimensional gel electrophoresis. By 6-24 h after intraocular injection of H/sub 3/(32)PO/sub 4/, approximately 20 optic nerve proteins ranging in size from 19 to 180 kilodaltons and in pI from 4.4 to 6.8 were seen to have incorporated radiolabel. Five of these proteins showed a robust increase in incorporation of phosphate during regeneration. Among the latter was an acidic (pI 4.5) 45-kilodalton protein, which has previously been shown to be conveyed by fast axonal transport and to increase dramatically in its rate of synthesis during regeneration of goldfish optic axons.

  4. Neurofilament protein synthesis in DRG neurons decreases more after peripheral axotomy than after central axotomy

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    Greenberg, S.G.; Lasek, R.J.

    1988-01-01

    Cytoskeletal protein synthesis was studied in DRG neurons after transecting either their peripheral or their central branch axons. Specifically, the axons were transected 5-10 mm from the lumbar-5 ganglion on one side of the animal; the DRGs from the transected side and contralateral control side were labeled with radiolabeled amino acids in vitro; radiolabeled proteins were separated by 2-dimensional (2D) PAGE; and the amounts of radiolabel in certain proteins of the experimental and control ganglia were quantified and compared. We focused on the neurofilament proteins because they are neuron-specific. If either the peripheral or central axons were cut, the amounts of radiolabeled neurofilament protein synthesized by the DRG neurons decreased between 1 and 10 d after transection. Neurofilament protein labeling decreased more after transection of the peripheral axons than after transection of the central axons. In contrast to axonal transections, sham operations or heat shock did not decrease the radiolabeling of the neurofilament proteins, and these procedures also affected the labeling of actin, tubulin, and the heat-shock proteins differently from transection. These results and others indicate that axonal transection leads to specific changes in the synthesis of cytoskeletal proteins of DRG neurons, and that these changes differ from those produced by stress to the animal or ganglia. Studies of the changes in neurofilament protein synthesis from 1 to 40 d after axonal transection indicate that the amounts of radiolabeled neurofilament protein synthesis were decreased during axonal elongation, but that they returned toward control levels when the axons reached cells that stopped elongation

  5. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

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    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-07-25

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

  6. Mechanistic logic underlying the axonal transport of cytosolic proteins

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    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  7. Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors

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    Gould, R.M.; Pant, H.; Gainer, H.; Tytell, M.

    1983-05-01

    The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. /sup 32/P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while (2-/sup 3/H)myo-inositol and L-(/sup 3/H(G))serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. (2-/sup 3/H)glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H)choline and (1-/sup 3/H)ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.

  8. Dynamic Changes of Neuroskeletal Proteins in DRGs Underlie Impaired Axonal Maturation and Progressive Axonal Degeneration in Type 1 Diabetes

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    Hideki Kamiya

    2009-01-01

    Full Text Available We investigated mechanisms underlying progressive axonal dysfunction and structural deficits in type 1 BB/Wor-rats from 1 week to 10 month diabetes duration. Motor and sensory conduction velocities were decreased after 4 and 6 weeks of diabetes and declined further over the remaining 9 months. Myelinated sural nerve fibers showed progressive deficits in fiber numbers and sizes. Structural deficits in unmyelinated axonal size were evident at 2 month and deficits in number were present at 4 mo. These changes were preceded by decreased availability of insulin, C-peptide and IGF-1 and decreased expression of neurofilaments and β-III-tubulin. Upregulation of phosphorylating stress kinases like Cdk5, p-GSK-3β, and p42/44 resulted in increased phosphorylation of neurofilaments. Increasing activity of p-GSK-3β correlated with increasing phosphorylation of NFH, whereas decreasing Cdk5 correlated with diminishing phosphorylation of NFM. The data suggest that impaired neurotrophic support results in sequentially impaired synthesis and postranslational modifications of neuroskeletal proteins, resulting in progressive deficits in axonal function, maturation and size.

  9. Fast axonal transport of 3H-leucin-labelled proteins in the unhurt and isolated optical nerve of rats

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    Wagner, H.E.

    1981-01-01

    The distribution of radioactivity of amino acid molecules incorporated in protein after injection of 3 H-Leucin into the right bulb was investigated and determined along optical nerve after 1, 2, and 4 h. A slightly increased radioactivity at the point of entrance of the optical nerves into the optical duct was found. A slightly reduced axon diameter was discussed as a possible cause. The radioactivity brought into the optical nerve via the vascular system was determined by measuring the contralateral optical nerve. In relation to the axonally transported activity, it was low. The speed of the fast axonal transport is 168 mm/d. If the processes ruling the amino acids in the perikaryon are taken into consideration, the transport speed is 240 mm/d. The application of the protein synthesis prohibitor, Cycloheximide, 5 minutes after the injection of Leucinin completely prevented the appearance of axonally transported labelled proteins. When cycloheximide was administered 2 h after Leucin, a significantly loner radioactivity than in the nerve could be determined after another 2 h; i.e. the incorporation of Leucin was not completed yet after 2 h. The profile of active compounds was the same as in the control group. In other experiments, the axonal transport of labelled proteins in isolated optical nerve fibres was tested. If the separation was carried out 2 h after the injection of Leucin an extreme reduction in activity could be determined after 1 or 2 h. The continued distribution of activity after cycloheximide treatment and removal of perikarya in comparison with the control indicate the continuation of the transport, also after separation of the axon from the perikaryon. This means that, during the time of the experiment, the mechanism of the fast axonal transport functions independently of the perikaryon. (orig./MG) [de

  10. Functional complexity of the axonal growth cone: a proteomic analysis.

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    Adriana Estrada-Bernal

    Full Text Available The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥ 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions.

  11. Glia to axon RNA transfer.

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    Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

    2014-03-01

    The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased. Copyright © 2013 Wiley Periodicals, Inc.

  12. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

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    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  13. Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth

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    Hai Li

    2016-07-01

    Full Text Available Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT and geranylgeranyl transferase I (PGGT-1. Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS. Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans.

  14. Specific effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

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    Shu Tang

    2016-01-01

    Full Text Available c-Jun NH2-terminal kinase (JNK-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These findings confirm that JNK-interacting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites.

  15. The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

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    Godzik, Katharina; Coleman, Michael P

    2015-04-01

    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

  16. Neuronal response of the hippocampal formation to injury: blood flow, glucose metabolism, and protein synthesis

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    Kameyama, M.; Wasterlain, C.G.; Ackermann, R.F.; Finch, D.; Lear, J.; Kuhl, D.E.

    1983-01-01

    The reaction of the hippocampal formation to entorhinal lesions was studied from the viewpoints of cerebral blood flow ([ 123 I]isopropyl-iodoamphetamine[IMP])-glucose utilization ([ 14 C]2-deoxyglucose), and protein synthesis ([ 14 C]leucine), using single- and double-label autoradiography. Researchers' studies showed decreased glucose utilization in the inner part, and increased glucose utilization in the outer part of the molecular layer of the dentate gyrus, starting 3 days after the lesion; increased uptake of [ 123 I]IMP around the lesion from 1 to 3 days postlesion; and starting 3 days after the lesion, marked decrease in [ 14 C]leucine incorporation into proteins and cell loss in the dorsal CA1 and dorsal subiculum in about one-half of the rats. These changes were present only in animals with lesions which invaded the ventral hippocampal formation in which axons of CA1 cells travel. By contrast, transsection of the 3rd and 4th cranial nerves resulted, 3 to 9 days after injury, in a striking increase in protein synthesis in the oculomotor and trochlear nuclei. These results raise the possibility that in some neurons the failure of central regeneration may result from the cell's inability to increase its rate of protein synthesis in response to axonal injury

  17. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

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    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  18. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.

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    Leite, Sérgio Carvalho; Sampaio, Paula; Sousa, Vera Filipe; Nogueira-Rodrigues, Joana; Pinto-Costa, Rita; Peters, Luanne Laurel; Brites, Pedro; Sousa, Mónica Mendes

    2016-04-19

    The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

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    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  20. Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats

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    Jasmin, B.J.; Lavoie, P.A.; Gardiner, P.F.

    1988-01-01

    In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins

  1. Na(v)1.8 channelopathy in mutant mice deficient for myelin protein zero is detrimental to motor axons

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    Moldovan, Mihai; Alvarez Herrero, Susana; Pinchenko, Volodymyr

    2011-01-01

    Myelin protein zero mutations were found to produce Charcot-Marie-Tooth disease phenotypes with various degrees of myelin impairment and axonal loss, ranging from the mild 'demyelinating' adult form to severe and early onset forms. Protein zero deficient homozygous mice ( ) show a severe and prog......Myelin protein zero mutations were found to produce Charcot-Marie-Tooth disease phenotypes with various degrees of myelin impairment and axonal loss, ranging from the mild 'demyelinating' adult form to severe and early onset forms. Protein zero deficient homozygous mice ( ) show a severe...... and progressive dysmyelinating neuropathy from birth with compromised myelin compaction, hypomyelination and distal axonal degeneration. A previous study using immunofluorescence showed that motor nerves deficient of myelin protein zero upregulate the Na(V)1.8 voltage gated sodium channel isoform, which...... is normally present only in restricted populations of sensory axons. The aim of this study was to investigate the function of motor axons in protein zero-deficient mice with particular emphasis on ectopic Na(V)1.8 voltage gated sodium channel. We combined 'threshold tracking' excitability studies...

  2. Direct transfer of viral and cellular proteins from varicella-zoster virus-infected non-neuronal cells to human axons.

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    Grigoryan, Sergei; Yee, Michael B; Glick, Yair; Gerber, Doron; Kepten, Eldad; Garini, Yuval; Yang, In Hong; Kinchington, Paul R; Goldstein, Ronald S

    2015-01-01

    Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.

  3. Electron microscopic localization of 3H-leucine in the neurons of the hypoglossal nerve during axonal reaction

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    Gylybov, G.P.; Chuchkov, Ch.Kh.; Davidov, M.S.

    1978-01-01

    The uptake of tritium-labelled leucine in the neuronal organelles with the aim of a follow-up of the dynamics in the protein synthesis in the motoneurons affected during axonal reaction was investigated. The experiments were carried out with rats, of which one of the hypoglossal nerve was crushed and the other was left intact. The labelled amino-acid was injected in the lateral cerebral ventricle 30 to 40 min before the sacrificing of each animal. The examination of the histological preparations shows that the neurons of the hypoglossal nerve cumulate to a larger extent the labelled precursor in comparison with the neuroglia. The perinuclear region, the nucleus, the nucleolus and the axonal hillock show preponderance in the accumulation. The activity greatly decreases at the more remote parts of the axon. The electron=microscopic data confirm these results and supplement them by exactly determining the localization of the labels in the individual organelles. The highest activity was found in the mitochondria, in the Golgi apparatus and in the lysosomes. This can be viewed as the result of intensified transfer of proteins from the ribosomes toward these organelles. There is, however, another possibility - the directly elevated biosynthesis. The elevated activity of the protein synthesis in the cell organelles, assume the authors, is related not only to preserving their structural proteins but also to intensifying axonal transport. (A.B.)

  4. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

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    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  5. Nociceptive DRG neurons express muscle lim protein upon axonal injury.

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    Levin, Evgeny; Andreadaki, Anastasia; Gobrecht, Philipp; Bosse, Frank; Fischer, Dietmar

    2017-04-04

    Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

  6. Ascending Midbrain Dopaminergic Axons Require Descending GAD65 Axon Fascicles for Normal Pathfinding

    Directory of Open Access Journals (Sweden)

    Claudia Marcela Garcia-Peña

    2014-06-01

    Full Text Available The Nigrostriatal pathway (NSP is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

  7. Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons.

    Science.gov (United States)

    Karten, Barbara; Vance, Dennis E; Campenot, Robert B; Vance, Jean E

    2003-02-07

    Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.

  8. Serotonin induces memory-like, rapamycin-sensitive hyperexcitability in sensory axons of aplysia that contributes to injury responses.

    Science.gov (United States)

    Weragoda, Ramal M S; Walters, Edgar T

    2007-09-01

    The induction of long-term facilitation (LTF) of synapses of Aplysia sensory neurons (SNs) by serotonin (5-HT) has provided an important mechanistic model of memory, but little is known about other long-term effects of 5-HT on sensory properties. Here we show that crushing peripheral nerves results in long-term hyperexcitability (LTH) of the axons of these nociceptive SNs that requires 5-HT activity in the injured nerve. Serotonin application to a nerve segment induces local axonal (but not somal) LTH that is inhibited by 5-HT-receptor antagonists. Blockade of crush-induced axonal LTH by an antagonist, methiothepin, provides evidence for mediation of this injury response by 5-HT. This is the first demonstration in any axon of neuromodulator-induced LTH, a phenomenon potentially important for long-lasting pain. Methiothepin does not reduce axonal LTH induced by local depolarization, so 5-HT is not required for all forms of axonal LTH. Serotonin-induced axonal LTH is expressed as reduced spike threshold and increased repetitive firing, whereas depolarization-induced LTH involves only reduced threshold. Like crush- and depolarization-induced LTH, 5-HT-induced LTH is blocked by inhibiting protein synthesis. Blockade by rapamycin, which also blocks synaptic LTF, is interesting because the eukaryotic protein kinase that is the target of rapamycin (TOR) has a conserved role in promoting growth by stimulating translation of proteins required for translation. Rapamycin sensitivity suggests that localized increases in translation of proteins that promote axonal conduction and excitability at sites of nerve injury may be regulated by the same signals that increase translation of proteins that promote neuronal growth.

  9. DISCO Interacting Protein 2 regulates axonal bifurcation and guidance of Drosophila mushroom body neurons.

    Science.gov (United States)

    Nitta, Yohei; Yamazaki, Daisuke; Sugie, Atsushi; Hiroi, Makoto; Tabata, Tetsuya

    2017-01-15

    Axonal branching is one of the key processes within the enormous complexity of the nervous system to enable a single neuron to send information to multiple targets. However, the molecular mechanisms that control branch formation are poorly understood. In particular, previous studies have rarely addressed the mechanisms underlying axonal bifurcation, in which axons form new branches via splitting of the growth cone. We demonstrate that DISCO Interacting Protein 2 (DIP2) is required for precise axonal bifurcation in Drosophila mushroom body (MB) neurons by suppressing ectopic bifurcation and regulating the guidance of sister axons. We also found that DIP2 localize to the plasma membrane. Domain function analysis revealed that the AMP-synthetase domains of DIP2 are essential for its function, which may involve exerting a catalytic activity that modifies fatty acids. Genetic analysis and subsequent biochemical analysis suggested that DIP2 is involved in the fatty acid metabolization of acyl-CoA. Taken together, our results reveal a function of DIP2 in the developing nervous system and provide a potential functional relationship between fatty acid metabolism and axon morphogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

    Science.gov (United States)

    Fréal, Amélie; Fassier, Coralie; Le Bras, Barbara; Bullier, Erika; De Gois, Stéphanie; Hazan, Jamilé; Hoogenraad, Casper C; Couraud, François

    2016-04-20

    The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity. Copyright © 2016 the authors 0270-6474/16/364421-13$15.00/0.

  11. Myelin-associated proteins labelled by slow axonal transport

    International Nuclear Information System (INIS)

    Giorgi, P.P.; DuBois, H.

    1981-01-01

    This paper deals with the problem of protein metabolism and provides evidence that the neuronal contribution to myelin metabolism may be restricted to lipids only. On the other hand this line of research led to the partial characterization of a group of neuronal proteins probably involved in axo-glial interactions subserving the onset of myelination and the structural maintenance of the mature myelin sheath. Intraocular injection of radioactive amino acids allows the study of the anterograde transport of labelled proteins along retinofugal fibres which are well myelinated. Myelin extracted from the optic nerve and tract under these conditions also contains labelled proteins. Three hypotheses are available to explain this phenomenon. To offer an explanation for this phenomenon the work was planned as follows. a) Characterization of the spatio-temporal pattern of labelling of myelin, in order to define the experimental conditions (survival time and region of the optic pathway to be studied) necessary to obtain maximal labelling. b) Characterization (by gel electrophoresis) of the myelin-associated proteins which become labelled by axonal transport, in order to work on a consistent pattern of labelling. c) Investigation of the possible mechanism responsible for the labelling of myelin-associated proteins. (Auth.)

  12. Aspartoacylase Deficiency in the White Matter of Human Immunodeficiency Virus Encephalitis: Novel Mechanism in Axonal Damage

    Directory of Open Access Journals (Sweden)

    Sankar Surendran

    2011-01-01

    Full Text Available Aspartoacylase/aminoacylase II (ASPA/ACY II is mainly synthesized in oligodendrocytes to contribute in myelin synthesis. Although axonal damage is seen in the brain with human immunodeficiency virus encephalitis (HIVE, ASPA contribution in the pathology is not known. Immunostaining study showed that ASPA protein is reduced in the white matter of patients with HIVE compared to the control. Western blot study further confirmed ASPA deficiency in the HIVE brain compared to the control. This paper suggests that HIVE condition affects ASPA to contribute in myelin loss/axonal damage seen in the disease.

  13. Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

    Science.gov (United States)

    Cioni, Jean-Michel; Wong, Hovy Ho-Wai; Bressan, Dario; Kodama, Lay; Harris, William A; Holt, Christine E

    2018-03-07

    The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Subacute ethanol consumption reverses p-xylene-induced decreases in axonal transport

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, S.; Lyerly, D.L.; Pope, C.N.

    1992-01-01

    Organic solvants, as a class, have been implicated as neurotoxic agents in humans and laboratory animals. The study was designed to assess the interaction between subacute ingestion of moderate levels of ethanol and the p-xylene-induced decreases in protein and glycoprotein synthesis and axonal transport in the rat optic system. The results indicated that animals maintained on 10% ethanol as a drinking liquid show less p-xylene-induced neurotoxicity than animals receiving no ethanol supplement.

  15. Meninges-derived cues control axon guidance.

    Science.gov (United States)

    Suter, Tracey A C S; DeLoughery, Zachary J; Jaworski, Alexander

    2017-10-01

    The axons of developing neurons travel long distances along stereotyped pathways under the direction of extracellular cues sensed by the axonal growth cone. Guidance cues are either secreted proteins that diffuse freely or bind the extracellular matrix, or membrane-anchored proteins. Different populations of axons express distinct sets of receptors for guidance cues, which results in differential responses to specific ligands. The full repertoire of axon guidance cues and receptors and the identity of the tissues producing these cues remain to be elucidated. The meninges are connective tissue layers enveloping the vertebrate brain and spinal cord that serve to protect the central nervous system (CNS). The meninges also instruct nervous system development by regulating the generation and migration of neural progenitors, but it has not been determined whether they help guide axons to their targets. Here, we investigate a possible role for the meninges in neuronal wiring. Using mouse neural tissue explants, we show that developing spinal cord meninges produce secreted attractive and repulsive cues that can guide multiple types of axons in vitro. We find that motor and sensory neurons, which project axons across the CNS-peripheral nervous system (PNS) boundary, are attracted by meninges. Conversely, axons of both ipsi- and contralaterally projecting dorsal spinal cord interneurons are repelled by meninges. The responses of these axonal populations to the meninges are consistent with their trajectories relative to meninges in vivo, suggesting that meningeal guidance factors contribute to nervous system wiring and control which axons are able to traverse the CNS-PNS boundary. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. An in vitro study of peptide-loaded alginate nanospheres for antagonizing the inhibitory effect of Nogo-A protein on axonal growth

    International Nuclear Information System (INIS)

    Zhai, Peng; Chen, X B; Schreyer, David J

    2015-01-01

    The adult mammalian central nervous system has limited ability to regenerate after injury. This is due, in part, to the presence of myelin-associated axon growth inhibitory proteins such as Nogo-A that bind and activate the Nogo receptor, leading to profound inhibition of actin-based motility within the growing axon tip. This paper presents an in vitro study of the use of a Nogo receptor-blocking peptide to antagonize the inhibitory effect of Nogo-A on axon growth. Alginate nanospheres were fabricated using an emulsion technique and loaded with Nogo receptor-blocking peptide, or with other model proteins. Protein release profiles were studied, and retention of the bioactivity of released proteins was verified. Primary dorsal root ganglion neurons were cultured and their ability to grow neurites was challenged with Nogo-A chimeric protein in the absence or presence of Nogo receptor antagonist peptide-loaded alginate nanospheres. Our results demonstrate that peptide released from alginate nanospheres could overcome the growth inhibitory effect of Nogo-A, suggesting that a similar peptide delivery strategy using alginate nanospheres might be used to improve axon regeneration within the injured central nervous system. (paper)

  17. Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy

    DEFF Research Database (Denmark)

    Fenrich, Keith K; Skelton, Nicole; MacDermid, Victoria E

    2007-01-01

    Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury......, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin...... at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein...

  18. Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

    Science.gov (United States)

    Chang, Jaerak; Blackstone, Craig

    2014-01-01

    Hereditary spastic paraplegias (HSPs) are a genetically diverse group of inherited neurological disorders (SPG1-72) with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the SPG4 gene product spastin. This is an ATPase associated with diverse cellular activities (AAA) protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1). Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development. PMID:25390646

  19. Spastin-interacting protein NA14/SSNA1 functions in cytokinesis and axon development.

    Directory of Open Access Journals (Sweden)

    Uma Goyal

    Full Text Available Hereditary spastic paraplegias (HSPs are a genetically diverse group of inherited neurological disorders (SPG1-72 with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the SPG4 gene product spastin. This is an ATPase associated with diverse cellular activities (AAA protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1. Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development.

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

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    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.

  1. 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.

  2. Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-05-05

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  3. Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-06-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  4. Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

    Science.gov (United States)

    Hancock, Melissa L.; Canetta, Sarah E.; Role, Lorna W.; Talmage, David A.

    2008-01-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function. PMID:18458158

  5. Formation of longitudinal axon pathways in Caenorhabditis elegans.

    Science.gov (United States)

    Hutter, Harald

    2017-11-18

    The small number of neurons and the simple architecture of the Caenorhabditis elegans (C. elegans) nervous system enables researchers to study axonal pathfinding at the level of individually identified axons. Axons in C. elegans extend predominantly along one of the two major body axes, the anterior-posterior axis and the dorso-ventral axis. This review will focus on axon navigation along the anterior-posterior axis, leading to the establishment of the longitudinal axon tracts, with a focus on the largest longitudinal axon tract, the ventral nerve cord (VNC). In the VNC, axons grow out in a stereotypic order, with early outgrowing axons (pioneers) playing an important role in guiding later outgrowing (follower) axons. Genetic screens have identified a number of genes specifically affecting the formation of longitudinal axon tracts. These genes include secreted proteins, putative receptors and adhesion molecules, as well as intracellular proteins regulating the cell's response to guidance cues. In contrast to dorso-ventral navigation, no major general guidance cues required for the establishment of longitudinal pathways have been identified so far. The limited penetrance of defects found in many mutants affecting longitudinal navigation suggests that guidance cues act redundantly in this process. The majority of the axon guidance genes identified in C. elegans are evolutionary conserved, i.e. have homologs in other animals, including vertebrates. For a number of these genes, a role in axon guidance has not been described outside C. elegans. Taken together, studies in C. elegans contribute to a fundamental understanding of the molecular basis of axonal navigation that can be extended to other animals, including vertebrates and probably humans as well. Copyright © 2017. Published by Elsevier Ltd.

  6. Structure and Function of an Actin-Based Filter in the Proximal Axon

    Directory of Open Access Journals (Sweden)

    Varuzhan Balasanyan

    2017-12-01

    Full Text Available Summary: The essential organization of microtubules within neurons has been described; however, less is known about how neuronal actin is arranged and the functional implications of its arrangement. Here, we describe, in live cells, an actin-based structure in the proximal axon that selectively prevents some proteins from entering the axon while allowing the passage of others. Concentrated patches of actin in proximal axons are present shortly after axonal specification in rat and zebrafish neurons imaged live, and they mark positions where anterogradely traveling vesicles carrying dendritic proteins halt and reverse. Patches colocalize with the ARP2/3 complex, and when ARP2/3-mediated nucleation is blocked, a dendritic protein mislocalizes to the axon. Patches are highly dynamic, with few persisting longer than 30 min. In neurons in culture and in vivo, actin appears to form a contiguous, semipermeable barrier, despite its apparently sparse distribution, preventing axonal localization of constitutively active myosin Va but not myosin VI. : Balasanyan et al. find dynamic patches of actin in proximal axons of live neurons, mature and newly differentiated, in culture and in vivo. Patches contribute to a filter that sequesters some proteins within the somatodendritic domain while allowing others to pass into the axon, leading to polarized localization of proteins.

  7. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

    Directory of Open Access Journals (Sweden)

    Martin Steuble

    2012-06-01

    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  8. Cargo distributions differentiate pathological axonal transport impairments.

    Science.gov (United States)

    Mitchell, Cassie S; Lee, Robert H

    2012-05-07

    Axonal transport is an essential process in neurons, analogous to shipping goods, by which energetic and cellular building supplies are carried downstream (anterogradely) and wastes are carried upstream (retrogradely) by molecular motors, which act as cargo porters. Impairments in axonal transport have been linked to devastating and often lethal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntington's, and Alzheimer's. Axonal transport impairment types include a decrease in available motors for cargo transport (motor depletion), the presence of defective or non-functional motors (motor dilution), and the presence of increased or larger cargos (protein aggregation). An impediment to potential treatment identification has been the inability to determine what type(s) of axonal transport impairment candidates that could be present in a given disease. In this study, we utilize a computational model and common axonal transport experimental metrics to reveal the axonal transport impairment general characteristics or "signatures" that result from three general defect types of motor depletion, motor dilution, and protein aggregation. Our results not only provide a means to discern these general impairments types, they also reveal key dynamic and emergent features of axonal transport, which potentially underlie multiple impairment types. The identified characteristics, as well as the analytical method, can be used to help elucidate the axonal transport impairments observed in experimental and clinical data. For example, using the model-predicted defect signatures, we identify the defect candidates, which are most likely to be responsible for the axonal transport impairments in the G93A SOD1 mouse model of ALS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Axonal Membranes and Their Domains: Assembly and Function of the Axon Initial Segment and Node of Ranvier

    Directory of Open Access Journals (Sweden)

    Andrew D. Nelson

    2017-05-01

    Full Text Available Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of

  10. GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

    Science.gov (United States)

    Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

    2011-01-01

    Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

  11. Acute nutritional axonal neuropathy.

    Science.gov (United States)

    Hamel, Johanna; Logigian, Eric L

    2018-01-01

    This study describes clinical, laboratory, and electrodiagnostic features of a severe acute axonal polyneuropathy common to patients with acute nutritional deficiency in the setting of alcoholism, bariatric surgery (BS), or anorexia. Retrospective analysis of clinical, electrodiagnostic, and laboratory data of patients with acute axonal neuropathy. Thirteen patients were identified with a severe, painful, sensory or sensorimotor axonal polyneuropathy that developed over 2-12 weeks with sensory ataxia, areflexia, variable muscle weakness, poor nutritional status, and weight loss, often with prolonged vomiting and normal cerebrospinal fluid protein. Vitamin B6 was low in half and thiamine was low in all patients when obtained before supplementation. Patients improved with weight gain and vitamin supplementation, with motor greater than sensory recovery. We suggest that acute or subacute axonal neuropathy in patients with weight loss or vomiting associated with alcohol abuse, BS, or dietary deficiency is one syndrome, caused by micronutrient deficiencies. Muscle Nerve 57: 33-39, 2018. © 2017 Wiley Periodicals, Inc.

  12. Mouse Intermittent Hypoxia Mimicking Apnea of Prematurity: Effects on Myelinogenesis and Axonal Maturation

    Science.gov (United States)

    CAI, JUN; TUONG, CHI MINH; ZHANG, YIPING; SHIELDS, CHRISTOPHER B.; GUO, GANG; FU, HUI; GOZAL, DAVID

    2014-01-01

    Premature babies are at high risk for both infantile apnea and long-term neurobehavioral deficits. Recent studies suggest that diffuse structural changes in brain white matter are a positive predictor of poor cognitive outcomes. Since oligodendrocyte maturation, myelination, axon development and synapse formation mainly occur in the 3rd trimester of gestation and 1st postnatal year, infantile apnea could lead to and/or exaggerate white matter impairments in preterm neonates. Therefore, we investigated oligodendroglia and axon development in a neonatal mouse model of intermittent hypoxia between postnatal days 2 to 10. During critical phases of central nervous system development, intermittent hypoxia induced hypomyelination in the corpus callosum, striatum, fornix and cerebellum, but not the pons or spinal cord. Intermittent hypoxia-elicited alterations in myelin-forming processes were reflected by decreased expression of myelin proteins, including MBP, PLP, MAG and CNPase, possibly due to arrested maturation of oligodendrocytes. Ultra-structural abnormalities were apparent in the myelin sheath and axon. Immature oligodendrocytes were more vulnerable to neonatal intermittent hypoxia exposures than developing axons, suggesting that hypomyelination may contribute, at least partially, to axonal deficits. Insufficient neurofilament synthesis with anomalous components of neurofilament subunits, β-tubulin and MAP2 isoforms indicated immaturity of axons in intermittent hypoxia-exposed mouse brains. In addition, down-regulation of Synapsin I, Synaptophysin and Gap-43 phosphorylation suggested a potential stunt in axonogenesis and synaptogenesis. The region-selective and complex impairment in brain white matter induced by intermittent hypoxia was further associated with electrophysiological changes that may underlie long-term neurobehavioral sequelae. PMID:21953180

  13. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

    Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

  14. RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

    Science.gov (United States)

    Grill, Brock; Chen, Lizhen; Tulgren, Erik D; Baker, Scott T; Bienvenut, Willy; Anderson, Matthew; Quadroni, Manfredo; Jin, Yishi; Garner, Craig C

    2012-02-22

    Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

  15. Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish

    OpenAIRE

    Stürmer, Claudia

    2011-01-01

    The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid...

  16. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

    Science.gov (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

    2017-01-01

    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  17. Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase

    International Nuclear Information System (INIS)

    Yang Dongren; Howard, Angela; Bruun, Donald; Ajua-Alemanj, Mispa; Pickart, Cecile; Lein, Pamela J.

    2008-01-01

    A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE -/- ) versus wild type (AChE +/+ ) mice indicated that while these OPs inhibited axonal growth in AChE +/+ DRG neurons, they had no effect on axonal growth in AChE -/- DRG neurons. However, transfection of AChE -/- DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs

  18. The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.

    Science.gov (United States)

    Florica, Roxana Oriana; Hipolito, Victoria; Bautista, Stephen; Anvari, Homa; Rapp, Chloe; El-Rass, Suzan; Asgharian, Alimohammad; Antonescu, Costin N; Killeen, Marie T

    2017-10-01

    The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor UNC-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or UNC-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or UNC-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either UNC-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Caenorhabditis elegans VEM-1, a novel membrane protein, regulates the guidance of ventral nerve cord-associated axons.

    Science.gov (United States)

    Runko, Erik; Kaprielian, Zaven

    2004-10-13

    In the developing CNS, pathfinding growth cones use intermediate target- and pioneer axon-associated guidance cues to navigate along stereotypical trajectories. We previously showed that the novel membrane-associated protein Vema is localized to the floor plate and the optic chiasm, intermediate targets located at the ventral midline of the spinal cord and diencephalon in the developing rodent CNS, respectively. Here, we report that the Caenorhabditis elegans ortholog of vema, vem-1, is expressed by the AVG pioneer midline neuron and by several neurons that extend longitudinally projecting axons into the ventral nerve cord (VNC). In vem-1 mutants and vem-1 (RNAi) animals, a subset of posteriorly projecting interneuron axons either fail to extend ventrally to the VNC and, instead, assume aberrant lateral positions or are inappropriately located in the left tract of the VNC. In addition, ventral motor neuron axons exhibit pathfinding errors within the VNC and along the dorsoventral body axis. The conserved UNC-40/DCC and SAX-3-/Robo receptors mediate signaling events that regulate axon guidance in a wide variety of systems. Double-mutant analyses reveal that vem-1 genetically interacts with unc-40 and is likely to function in parallel with sax-3 to regulate the guidance of a subset of VNC-associated interneuron and motor neuron axons. Consistent with these genetic data, we also show that VEM-1 is capable of physically interacting with UNC-40 but not SAX-3.

  20. Axon-glia interaction and membrane traffic in myelin formation

    Directory of Open Access Journals (Sweden)

    Robin eWhite

    2014-01-01

    Full Text Available In vertebrate nervous systems myelination of neuronal axons has evolved to increase conduction velocity of electrical impulses with minimal space and energy requirements. Myelin is formed by specialised glial cells which ensheath axons with a lipid-rich insulating membrane. Myelination is a multi-step process initiated by axon-glia recognition triggering glial polarisation followed by targeted myelin membrane expansion and compaction. Thereby, a myelin sheath of complex subdomain structure is established. Continuous communication between neurons and glial cells is essential for myelin maintenance and axonal integrity. A diverse group of diseases, from multiple sclerosis to schizophrenia, have been linked to malfunction of myelinating cells reflecting the physiological importance of the axon-glial unit. This review describes the mechanisms of axonal signal integration by oligodendrocytes emphasising the central role of the Src-family kinase Fyn during CNS myelination. Furthermore, we discuss myelin membrane trafficking with particular focus on endocytic recycling and the control of PLP (proteolipid protein transport by SNARE proteins. Finally, PLP mistrafficking is considered in the context of myelin diseases.

  1. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

    Science.gov (United States)

    Sasaki, Yo; Nakagawa, Takashi; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

    2016-01-01

    Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.001 PMID:27735788

  2. Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons

    DEFF Research Database (Denmark)

    Edbladh, M; Svenningsen, Åsa Fex; Ekström, P A

    1994-01-01

    We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological...... dose (10 ng/ml, approximately 2 nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin...... increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner...

  3. Axonal sprouting regulates myelin basic protein gene expression in denervated mouse hippocampus

    DEFF Research Database (Denmark)

    Jensen, M B; Poulsen, F R; Finsen, B

    2000-01-01

    to 35 days after transection of the entorhino-hippocampal perforant path axonal projection. In situ hybridization analysis showed that anterograde axonal and terminal degeneration lead to upregulated oligodendrocyte MBP mRNA expression starting between day 2 and day 4, in (1) the deep part of stratum...... axonal and terminal degeneration, myelin degenerative changes, microglial activation and axotomi-induced axonal sprouting. Oligodendrocyte MBP mRNA expression reached maximum in both these areas at day 7. MBP gene transcription remained constant in stratum radiatum, stratum pyramidale and stratum oriens...... of CA1, areas that were unaffected by perforant path transection. These results provide strong evidence that oligodendrocyte MBP gene expression can be regulated by axonal sprouting independently of microglial activation in the injured adult CNS....

  4. Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

    Science.gov (United States)

    Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu; Yomo, Tetsuya

    2014-11-01

    In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Modulation of protein synthesis by polyamines.

    Science.gov (United States)

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis. © 2015 International Union of Biochemistry and Molecular Biology.

  6. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    Science.gov (United States)

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  7. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    2016-06-01

    Full Text Available Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

  8. Noninvasive Detection and Differentiation of Axonal Injury/Loss, Demyelination, and Inflammation

    Science.gov (United States)

    2014-10-01

    phosphorylated neurofilament primary antibody (SMI-31; 1:1000, Covance , US) to stain non-injured axons, and in rabbit anti-myelin basic protein (MBP) primary...neurofilament antibody (SMI- 31; 1:1000, Covance , US) to stain non-injured axons or with rabbit anti-myelin basic protein (MBP) antibody (1:1000, Sigma Inc

  9. N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

    Science.gov (United States)

    Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

    2016-02-01

    Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

  10. Protein intake does not increase vastus lateralis muscle protein synthesis during cycling

    DEFF Research Database (Denmark)

    Hulston, CJ; Wolsk, Emil; Grøndahl, Thomas Sahl

    2011-01-01

    PURPOSE: This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. METHODS: Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution...... sampling, and blood flow measurements. Muscle protein synthesis was calculated from the incorporation of l-[ring-C6]phenylalanine into protein. RESULTS: Consuming protein during exercise increased leg protein synthesis and decreased net leg protein breakdown; however, protein ingestion did not increase...... protein synthesis within the highly active vastus lateralis muscle (0.029%·h(-1), ± 0.004%·h(-1), and 0.030%·h(-1), ± 0.003%·h(-1), in CHO and CHO + P, respectively; P = 0.88). In contrast, consuming protein, during exercise and recovery, increased postexercise vastus lateralis muscle protein synthesis...

  11. Noncovalent synthesis of protein dendrimers

    NARCIS (Netherlands)

    Lempens, E.H.M.; Baal, van I.; Dongen, van J.L.J.; Hackeng, T.M.; Merkx, M.; Meijer, E.W.

    2009-01-01

    The covalent synthesis of complex biomolecular systems such as multivalent protein dendrimers often proceeds with low efficiency, thereby making alternative strategies based on noncovalent chemistry of high interest. Here, the synthesis of protein dendrimers using a strong but noncovalent

  12. Characterization of axon formation in the embryonic stem cell-derived motoneuron.

    Science.gov (United States)

    Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

    2011-01-01

    The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

  13. Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

    International Nuclear Information System (INIS)

    Kefalides, N.A.

    1986-01-01

    The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

  14. TIPsy tour guides: How microtubule plus-end tracking proteins (+TIPs facilitate axon guidance

    Directory of Open Access Journals (Sweden)

    Elizabeth A Bearce

    2015-06-01

    Full Text Available The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone’s distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules in growth cone navigation. Here, we focus on the role of singular pioneer microtubules, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs. These +TIPs accumulate at the dynamic ends of microtubules, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events.

  15. Leucine stimulation of skeletal muscle protein synthesis

    International Nuclear Information System (INIS)

    Layman, D.K.; Grogan, C.K.

    1986-01-01

    Previous work in this laboratory has demonstrated a stimulatory effect of leucine on skeletal muscle protein synthesis measured in vitro during catabolic conditions. Studies in other laboratories have consistently found this effect in diaphragm muscle, however, studies examining effects on nitrogen balance or with in vivo protein synthesis in skeletal muscle are equivocal. This experiment was designed to determine the potential of leucine to stimulate skeletal muscle protein synthesis in vivo. Male Sprague-Dawley rats weighing 200 g were fasted for 12 hrs, anesthetized, a jugular cannula inserted, and protein synthesis measured using a primed continuous infusion of 14 C-tyrosine. A plateau in specific activity was reached after 30 to 60 min and maintained for 3 hrs. The leucine dose consisted of a 240 umole priming dose followed by a continuous infusion of 160 umoles/hr. Leucine infusion stimulated protein synthesis in the soleus muscle (28%) and in the red (28%) and white portions (12%) of the gastrocnemius muscle compared with controls infused with only tyrosine. The increased rates of protein synthesis were due to increased incorporation of tyrosine into protein and to decreased specific activity of the free tyrosine pool. These data indicate that infusion of leucine has the potential to stimulate in vivo protein synthesis in skeletal muscles

  16. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  17. Protein synthesis in the growing rat lung

    International Nuclear Information System (INIS)

    Kelley, J.; Chrin, L.

    1986-01-01

    Developmental control of protein synthesis in the postnatal growth of the lung has not been systematically studied. In male Fischer 344 rats, lung growth continues linearly as a function of body weight (from 75 to 450 g body weight). To study total protein synthesis in lungs of growing rats, we used the technique of constant intravenous infusion of tritiated leucine, an essential amino acid. Lungs of sacrificed animals were used to determine the leucine incorporation rate into newly synthesized protein. The specific radioactivity of the leucine associated with tRNA extracted from the same lungs served as an absolute index of the precursor leucine pool used for lung protein synthesis. On the basis of these measurements, we were able to calculate the fractional synthesis rate (the proportion of total protein destroyed and replaced each day) of pulmonary proteins for each rat. Under the conditions of isotope infusion, leucyl-tRNA very rapidly equilibrates with free leucine of the plasma and of the extracellular space of the lung. Infusions lasting 30 minutes or less yielded linear rates of protein synthesis without evidence of contamination of lung proteins by newly labeled intravascular albumin. The fractional synthesis rate is considerably higher in juvenile animals (55% per day) than in adult rats (20% per day). After approximately 12 weeks of age, the fractional synthesis rate remains extremely constant in spite of continued slow growth of the lung. It is apparent from these data that in both young and adult rats the bulk of total protein synthesis is devoted to rapidly turning over proteins and that less than 4 percent of newly made protein is committed to tissue growth

  18. DISCO interacting protein 2 determines direction of axon projection under the regulation of c-Jun N-terminal kinase in the Drosophila mushroom body

    International Nuclear Information System (INIS)

    Nitta, Yohei; Sugie, Atsushi

    2017-01-01

    Precisely controlled axon guidance for complex neuronal wiring is essential for appropriate neuronal function. c-Jun N-terminal kinase (JNK) was found to play a role in axon guidance recently as well as in cell proliferation, protection and apoptosis. In spite of many genetic and molecular studies on these biological processes regulated by JNK, how JNK regulates axon guidance accurately has not been fully explained thus far. To address this question, we use the Drosophila mushroom body (MB) as a model since the α/β axons project in two distinct directions. Here we show that DISCO interacting protein 2 (DIP2) is required for the accurate direction of axonal guidance. DIP2 expression is under the regulation of Basket (Bsk), the Drosophila homologue of JNK. We additionally found that the Bsk/DIP2 pathway is independent from the AP-1 transcriptional factor complex pathway, which is directly activated by Bsk. In conclusion, our findings revealed DIP2 as a novel effector downstream of Bsk modulating the direction of axon projection. - Highlights: • DIP2 is required for accurate direction of axon guidance in Drosophila mushroom body. • DIP2 is a downstream of JNK in the axon guidance of Drosophila mushroom body neuron. • JNK/DIP2 pathway is independent from JNK/AP-1 transcriptional factor complex pathway.

  19. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    OpenAIRE

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential a...

  20. Independent signaling by Drosophila insulin receptor for axon guidance and growth

    Directory of Open Access Journals (Sweden)

    Caroline Rita Li

    2014-01-01

    Full Text Available The Drosophila insulin receptor (DInR regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin-receptor-substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock. In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail, important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock-binding sites were in separate portions of the C-tail from the previously identified Chico-binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth, and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all 5 NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. Mutation of these 5 NPXY motifs did not affect photoreceptor axon guidance, showing that different sites within DInR control growth and axon guidance.

  1. Brief electrical stimulation accelerates axon regeneration in the peripheral nervous system and promotes sensory axon regeneration in the central nervous system.

    Science.gov (United States)

    Gordon, Tessa; Udina, Esther; Verge, Valerie M K; de Chaves, Elena I Posse

    2009-10-01

    Injured peripheral but not central nerves regenerate their axons but functional recovery is often poor. We demonstrate that prolonged periods of axon separation from targets and Schwann cell denervation eliminate regenerative capacity in the peripheral nervous system (PNS). A substantial delay of 4 weeks for all regenerating axons to cross a site of repair of sectioned nerve contributes to the long period of separation. Findings that 1h 20Hz bipolar electrical stimulation accelerates axon outgrowth across the repair site and the downstream reinnervation of denervated muscles in rats and human patients, provides a new and exciting method to improve functional recovery after nerve injuries. Drugs that elevate neuronal cAMP and activate PKA promote axon outgrowth in vivo and in vitro, mimicking the electrical stimulation effect. Rapid expression of neurotrophic factors and their receptors and then of growth associated proteins thereafter via cAMP, is the likely mechanism by which electrical stimulation accelerates axon outgrowth from the site of injury in both peripheral and central nervous systems.

  2. Protein synthesis in geostimulated root caps

    Science.gov (United States)

    Feldman, L. J.

    1982-01-01

    A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

  3. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization.

    Science.gov (United States)

    Hummel, T; Leifker, K; Klämbt, C

    2000-04-01

    In Drosophila, the correct formation of the segmental commissures depends on neuron-glial interactions at the midline. The VUM midline neurons extend axons along which glial cells migrate in between anterior and posterior commissures. Here, we show that the gene kette is required for the normal projection of the VUM axons and subsequently disrupts glial migration. Axonal projection defects are also found for many other moto- and interneurons. In addition, kette affects the cell morphology of mesodermal and epidermal derivatives, which show an abnormal actin cytoskeleton. The KETTE protein is homologous to the transmembrane protein HEM-2/NAP1 evolutionary conserved from worms to vertebrates. In vitro analysis has shown a specific interaction of the vertebrate HEM-2/NAP1 with the SH2-SH3 adapter protein NCK and the small GTPase RAC1, which both have been implicated in regulating cytoskeleton organization and axonal growth. Hypomorphic kette mutations lead to axonal defects similar to mutations in the Drosophila NCK homolog dreadlocks. Furthermore, we show that kette and dock mutants genetically interact. NCK is thought to interact with the small G proteins RAC1 and CDC42, which play a role in axonal growth. In line with these observations, a kette phenocopy can be obtained following directed expression of mutant DCDC42 or DRAC1 in the CNS midline. In addition, the kette mutant phenotype can be partially rescued by expression of an activated DRAC1 transgene. Our data suggest an important role of the HEM-2 protein in cytoskeletal organization during axonal pathfinding.

  4. Protein synthesis controls phosphate homeostasis.

    Science.gov (United States)

    Pontes, Mauricio H; Groisman, Eduardo A

    2018-01-01

    Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium , this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg 2+ ), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg 2+ promotes an uptake in Mg 2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature. © 2018 Pontes and Groisman; Published by Cold Spring Harbor Laboratory Press.

  5. Formation of compact myelin is required for maturation of the axonal cytoskeleton

    Science.gov (United States)

    Brady, S. T.; Witt, A. S.; Kirkpatrick, L. L.; de Waegh, S. M.; Readhead, C.; Tu, P. H.; Lee, V. M.

    1999-01-01

    Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

  6. Chronological protein synthesis in regenerating rat liver.

    Science.gov (United States)

    He, Jinjun; Hao, Shuai; Zhang, Hao; Guo, Fuzheng; Huang, Lingyun; Xiao, Xueyuan; He, Dacheng

    2015-07-01

    Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Albumin synthesis in protein energy malnutrition

    Energy Technology Data Exchange (ETDEWEB)

    Duggan, C; Hardy, S; Kleinman, R E [Harvard Medical School, Boston, MA (United States); Lembcke, J [Instituto de Investigacion Nutricional, La Molina, Lima (Peru); Young, V E [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Lab. of Human Nutrition

    1994-12-31

    The dietary treatment of protein-energy malnutrition (PEM) has been designed on an empirical basis, with outcomes for successful management including body weight gain and resolution of apathy. We propose using the measurements of protein synthesis as a more objective measure of renourishment. We will therefore randomize a group of malnourished children (weigh-for-height Z score <-2.0) to receive either a standard (10% of calories as protein) or increased (15%) amount of dietary protein early in their recovery phase. We will calculate albumin synthesis rates via the flooding dose technique, using {sup 13}C-leucine and serial measurements of {sup 13}C-enrichment of albumin. Isotope infusions will be performed on days one and three, following a standard three hour fast. Since albumin synthesis is reduced under the influence of cytokines which mediate the inflammatory response, results will be stratified according to the presence or absence of clinically apparent infections. We hypothesize that the provision of added dietary protein will optimize albumin synthesis rates in PEM as well as attenuate the reduction in albumin synthesis seen in the presence of infections. (author). 20 refs.

  8. Synaptic Democracy and Vesicular Transport in Axons

    Science.gov (United States)

    Bressloff, Paul C.; Levien, Ethan

    2015-04-01

    Synaptic democracy concerns the general problem of how regions of an axon or dendrite far from the cell body (soma) of a neuron can play an effective role in neuronal function. For example, stimulated synapses far from the soma are unlikely to influence the firing of a neuron unless some sort of active dendritic processing occurs. Analogously, the motor-driven transport of newly synthesized proteins from the soma to presynaptic targets along the axon tends to favor the delivery of resources to proximal synapses. Both of these phenomena reflect fundamental limitations of transport processes based on a localized source. In this Letter, we show that a more democratic distribution of proteins along an axon can be achieved by making the transport process less efficient. This involves two components: bidirectional or "stop-and-go" motor transport (which can be modeled in terms of advection-diffusion), and reversible interactions between motor-cargo complexes and synaptic targets. Both of these features have recently been observed experimentally. Our model suggests that, just as in human societies, there needs to be a balance between "efficiency" and "equality".

  9. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

  10. Synthesis of Lipidated Proteins.

    Science.gov (United States)

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

  11. N-Propionylmannosamine stimulates axonal elongation in a murine model of sciatic nerve injury

    Directory of Open Access Journals (Sweden)

    Christian Witzel

    2015-01-01

    Full Text Available Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-acyl side chain. N-Propionylmannosamine (ManNProp increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp (200 mg/kg or saline solution 5 days before injury, and continued it until nerve harvest (5 days after transection. ManNProp significantly increased the mean distance of axonal regeneration (2.49 mm vs. 1.53 mm; P < 0.005 and the number of arborizing axons (21% vs. 16% P = 0.008 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.

  12. Slowing of axonal regeneration is correlated with increased axonal viscosity during aging

    Directory of Open Access Journals (Sweden)

    Heidemann Steven R

    2010-10-01

    Full Text Available Abstract Background As we age, the speed of axonal regeneration declines. At the biophysical level, why this occurs is not well understood. Results To investigate we first measured the rate of axonal elongation of sensory neurons cultured from neonatal and adult rats. We found that neonatal axons grew 40% faster than adult axons (11.5 µm/hour vs. 8.2 µm/hour. To determine how the mechanical properties of axons change during maturation, we used force calibrated towing needles to measure the viscosity (stiffness and strength of substrate adhesion of neonatal and adult sensory axons. We found no significant difference in the strength of adhesions, but did find that adult axons were 3 times intrinsically stiffer than neonatal axons. Conclusions Taken together, our results suggest decreasing axonal stiffness may be part of an effective strategy to accelerate the regeneration of axons in the adult peripheral nervous system.

  13. Independent signaling by Drosophila insulin receptor for axon guidance and growth.

    Science.gov (United States)

    Li, Caroline R; Guo, Dongyu; Pick, Leslie

    2013-01-01

    The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin receptor substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock binding sites were in separate portions of the C-tail from the previously identified Chico binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all five NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. These animals resembled chico mutants, supporting the notion that DInR interacts directly with Chico in vivo to control body size. Mutation of these five NPXY motifs did not affect photoreceptor axon guidance, segregating the roles of DInR in the

  14. Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation

    Science.gov (United States)

    Petralia, Ronald S.; Ott, Carolyn; Wang, Ya-Xian; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2015-01-01

    The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. PMID:26658865

  15. Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases.

    Science.gov (United States)

    Brady, Scott T; Morfini, Gerardo A

    2017-09-01

    Neurons affected in a wide variety of unrelated adult-onset neurodegenerative diseases (AONDs) typically exhibit a "dying back" pattern of degeneration, which is characterized by early deficits in synaptic function and neuritic pathology long before neuronal cell death. Consistent with this observation, multiple unrelated AONDs including Alzheimer's disease, Parkinson's disease, Huntington's disease, and several motor neuron diseases feature early alterations in kinase-based signaling pathways associated with deficits in axonal transport (AT), a complex cellular process involving multiple intracellular trafficking events powered by microtubule-based motor proteins. These pathogenic events have important therapeutic implications, suggesting that a focus on preservation of neuronal connections may be more effective to treat AONDs than addressing neuronal cell death. While the molecular mechanisms underlying AT abnormalities in AONDs are still being analyzed, evidence has accumulated linking those to a well-established pathological hallmark of multiple AONDs: altered patterns of neuronal protein phosphorylation. Here, we present a short overview on the biochemical heterogeneity of major motor proteins for AT, their regulation by protein kinases, and evidence revealing cell type-specific AT specializations. When considered together, these findings may help explain how independent pathogenic pathways can affect AT differentially in the context of each AOND. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation.

    Directory of Open Access Journals (Sweden)

    Qingyu Qin

    Full Text Available Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK and murine double minute (Mdm2 E3 ligase. Growth cone collapse induced by genetic (npc1-/- or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1-/- mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.

  17. Two Modes of the Axonal Interferon Response Limit Alphaherpesvirus Neuroinvasion

    Directory of Open Access Journals (Sweden)

    Ren Song

    2016-02-01

    Full Text Available Infection by alphaherpesviruses, including herpes simplex virus (HSV and pseudorabies virus (PRV, typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS. Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs. The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-β or gamma interferon (IFN-γ significantly diminished the number of herpes simplex virus 1 (HSV-1 and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-β induced STAT1 phosphorylation (p-STAT1 only in axons, while exposure of axons to IFN-γ induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-γ, but not those induced by IFN-β. Proteomic analysis of IFN-β- or IFN-γ-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-γ, IFN-β induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion.

  18. Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

    Directory of Open Access Journals (Sweden)

    Takehiro Nishikawa

    2012-01-01

    Full Text Available Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype and the protein translated from the information (phenotype, which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE system and a fluorescence-activated cell sorter (FACS used as a microcompartment, in vitro protein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized by in vitro protein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP and β-glucuronidase are described. We discuss the future directions for this method and its applications.

  19. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  20. SHORT-TERM MEMORY IS INDEPENDENT OF BRAIN PROTEIN SYNTHESIS

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Hasker P.; Rosenzweig, Mark R.; Jones, Oliver W.

    1980-09-01

    Male Swiss albino CD-1 mice given a single injection of a cerebral protein synthesis inhibitor, anisomycin (ANI) (1 mg/animal), 20 min prior to single trial passive avoidance training demonstrated impaired retention at tests given 3 hr, 6 hr, 1 day, and 7 days after training. Retention was not significantly different from saline controls when tests were given 0.5 or 1.5 hr after training. Prolonging inhibition of brain protein synthesis by giving either 1 or 2 additional injections of ANI 2 or 2 and 4 hr after training did not prolong short-term retention performance. The temporal development of impaired retention in ANI treated mice could not be accounted for by drug dosage, duration of protein synthesis inhibition, or nonspecific sickness at test. In contrast to the suggestion that protein synthesis inhibition prolongs short-term memory (Quinton, 1978), the results of this experiment indicate that short-term memory is not prolonged by antibiotic drugs that inhibit cerebral protein synthesis. All evidence seems consistent with the hypothesis that short-term memory is protein synthesis independent and that the establishment of long-term memory depends upon protein synthesis during or shortly after training. Evidence for a role of protein synthesis in memory maintenance is discussed.

  1. Structure and function of the contactin-associated protein family in myelinated axons and their relationship with nerve diseases

    Institute of Scientific and Technical Information of China (English)

    Yan Zou; De-en Xu; Wei-feng Zhang; Hai-ying Liu; Xia Li; Xing Zhang; Xiao-fang Ma; Yang Sun; Shi-yi Jiang; Quan-hong Ma

    2017-01-01

    The contactin-associated protein (Caspr) family participates in nerve excitation and conduction, and neurotransmitter release in myelinated axons. We analyzed the structures and functions of the Caspr family–CNTNAP1 (Caspr1), CNTNAP2 (Caspr2), CNTNAP3 (Caspr3), CNTNAP4 (Caspr4) and CNTNAP5 (Caspr5), Caspr1–5 is not only involved in the formation of myelinated axons, but also participates in maintaining the stability of adjacent connections. Caspr1 participates in the formation, differentiation, and proliferation of neurons and astrocytes, and in motor control and cognitive function. We also analyzed the relationship between the Caspr family and neurodegenerative diseases, multiple sclerosis, and autoimmune encephalitis. However, the effects of Caspr on disease course and prognosis remain poorly understood. The effects of Caspr on disease diagnosis and treatment need further investigation.

  2. Alterations in a Unique Class of Cortical Chandelier Cell Axon Cartridges in Schizophrenia.

    Science.gov (United States)

    Rocco, Brad R; DeDionisio, Adam M; Lewis, David A; Fish, Kenneth N

    2017-07-01

    The axons of chandelier cells (ChCs) target the axon initial segment of pyramidal neurons, forming an array of boutons termed a cartridge. In schizophrenia, the density of cartridges detectable by gamma-aminobutyric acid (GABA) membrane transporter 1 immunoreactivity is lower, whereas the density of axon initial segments detectable by immunoreactivity for the α2 subunit of the GABA A receptor is higher in layers 2/superficial 3 of the prefrontal cortex. These findings were interpreted as compensatory responses to lower GABA levels in ChCs. However, we recently found that in schizophrenia, ChC cartridge boutons contain normal levels of the 67 kDa isoform of glutamic acid decarboxylase (GAD67) protein, the enzyme responsible for GABA synthesis in these boutons. To understand these findings we quantified the densities of ChC cartridges immunoreactive for vesicular GABA transporter (vGAT+), which is present in all cartridge boutons, and the subset of cartridges that contain calbindin (CB+). Prefrontal cortex tissue sections from 20 matched pairs of schizophrenia and unaffected comparison subjects were immunolabeled for vGAT, GAD67, and CB. The mean density of vGAT+/CB+ cartridges was 2.7-fold higher, exclusively in layer 2 of schizophrenia subjects, whereas the density of vGAT+/CB- cartridges did not differ between subject groups. Neither vGAT, CB, or GAD67 protein levels per ChC bouton nor the number of boutons per cartridge differed between subject groups. Our findings of a greater density of CB+ ChC cartridges in prefrontal cortex layer 2 from schizophrenia subjects suggests that the normal developmental pruning of these cartridges is blunted in the illness. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  3. Dynamics of target recognition by interstitial axon branching along developing cortical axons.

    Science.gov (United States)

    Bastmeyer, M; O'Leary, D D

    1996-02-15

    Corticospinal axons innervate their midbrain, hindbrain, and spinal targets by extending collateral branches interstitially along their length. To establish that the axon shaft rather than the axonal growth cone is responsible for target recognition in this system, and to characterize the dynamics of interstitial branch formation, we have studied this process in an in vivo-like setting using slice cultures from neonatal mice containing the entire pathway of corticospinal axons. Corticospinal axons labeled with the dye 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (or Dil) were imaged using time-lapse video microscopy of their pathway overlying the basilar pons, their major hindbrain target. The axon shaft millimeters behind the growth cone exhibits several dynamic behaviors, including the de novo formation of varicosities and filopodia-like extensions, and a behavior that we term "pulsation," which is characterized by a variable thickening and thining of short segments of the axon. An individual axon can have multiple sites of branching activity, with many of the branches being transient. These dynamic behaviors occur along the portion of the axon shaft overlying the basilar pons, but not just caudal to it. Once the collaterals extend into the pontine neuropil, they branch further in the neuropil, while the parent axon becomes quiescent. Thus, the branching activity is spatially restricted to specific portions of the axon, as well as temporally restricted to a relatively brief time window. These findings provide definitive evidence that collateral branches form de novo along corticospinal axons and establish that the process of target recognition in this system is a property of the axon shaft rather than the leading growth cone.

  4. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    Science.gov (United States)

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  5. Protein synthesis in x-irradiated rabbit lens

    International Nuclear Information System (INIS)

    Garadi, R.; Foltyn, A.R.; Giblin, F.J.; Reddy, V.N.

    1984-01-01

    The present study deals with the incorporation of 35 S methionine into lens crystallins as a function of time after x-irradiation. Crystallin synthesis is first affected approximately 4 weeks following x-irradiation. This coincides with the time period at which the ratio of the two cations in the lens is affected, as shown in earlier studies. A greater decrease in 35 S-methionine incorporation into crystallins is observed between 5-7 weeks following x-irradiation in good agreement with a cation imbalance at these time intervals. These studies also revealed for the first time that the change in cation distribution can affect not only crystallin synthesis, but also the synthesis of certain polypeptides of lens membranes. No alteration in protein synthesis could be detected in lens epithelium even after 7 weeks following irradiation. In addition to the effect of Na+ and K+ levels on protein synthesis, an impaired transport of amino acids into the x-rayed lens was also found to be a factor in the observed reduction in synthesis of the crystallin, cytoskeletal and membrane proteins of the fiber cells. It is concluded that Na+/K+ ratio as well as the availability of amino acids in the lens are important factors in protein synthesis of x-ray cataracts

  6. Albumin synthesis in protein energy malnutrition

    International Nuclear Information System (INIS)

    Duggan, C.; Hardy, S.; Kleinman, R.E.; Lembcke, J.; Young, V.E.

    1994-01-01

    The dietary treatment of protein-energy malnutrition (PEM) has been designed on an empirical basis, with outcomes for successful management including body weight gain and resolution of apathy. We propose using the measurements of protein synthesis as a more objective measure of renourishment. We will therefore randomize a group of malnourished children (weigh-for-height Z score 13 C-leucine and serial measurements of 13 C-enrichment of albumin. Isotope infusions will be performed on days one and three, following a standard three hour fast. Since albumin synthesis is reduced under the influence of cytokines which mediate the inflammatory response, results will be stratified according to the presence or absence of clinically apparent infections. We hypothesize that the provision of added dietary protein will optimize albumin synthesis rates in PEM as well as attenuate the reduction in albumin synthesis seen in the presence of infections. (author). 20 refs

  7. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    Science.gov (United States)

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. © 2015 Wiley Periodicals, Inc.

  8. Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    Science.gov (United States)

    Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

    2014-11-01

    Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

  9. Axonal GABAA receptors.

    Science.gov (United States)

    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  10. The relationship between protein synthesis and protein degradation in object recognition memory.

    Science.gov (United States)

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Structure and function of the contactin-associated protein family in myelinated axons and their relationship with nerve diseases

    Directory of Open Access Journals (Sweden)

    Yan Zou

    2017-01-01

    Full Text Available The contactin-associated protein (Caspr family participates in nerve excitation and conduction, and neurotransmitter release in myelinated axons. We analyzed the structures and functions of the Caspr family–CNTNAP1 (Caspr1, CNTNAP2 (Caspr2, CNTNAP3 (Caspr3, CNTNAP4 (Caspr4 and CNTNAP5 (Caspr5, Caspr1–5 is not only involved in the formation of myelinated axons, but also participates in maintaining the stability of adjacent connections. Caspr1 participates in the formation, differentiation, and proliferation of neurons and astrocytes, and in motor control and cognitive function. We also analyzed the relationship between the Caspr family and neurodegenerative diseases, multiple sclerosis, and autoimmune encephalitis. However, the effects of Caspr on disease course and prognosis remain poorly understood. The effects of Caspr on disease diagnosis and treatment need further investigation.

  12. Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

    Science.gov (United States)

    Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W

    2016-06-01

    Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

  13. Effects of Whey, Caseinate, or Milk Protein Ingestion on Muscle Protein Synthesis after Exercise.

    Science.gov (United States)

    Kanda, Atsushi; Nakayama, Kyosuke; Sanbongi, Chiaki; Nagata, Masashi; Ikegami, Shuji; Itoh, Hiroyuki

    2016-06-03

    Whey protein (WP) is characterized as a "fast" protein and caseinate (CA) as a "slow" protein according to their digestion and absorption rates. We hypothesized that co-ingestion of milk proteins (WP and CA) may be effective for prolonging the muscle protein synthesis response compared to either protein alone. We therefore compared the effect of ingesting milk protein (MP) to either WP or CA alone on muscle protein synthesis after exercise in rats. We also compared the effects of these milk-derived proteins to a control, soy protein (SP). Male Sprague-Dawley rats swam for two hours. Immediately after exercise, one of the following four solutions was administered: WP, CA, MP, or SP. Individual rats were euthanized at designated postprandial time points and triceps muscle samples collected for measurement of the protein fractional synthesis rate (FSR). FSR tended to increase in all groups post-ingestion, although the initial peaks of FSR occurred at different times (WP, peak time = 60 min, FSR = 7.76%/day; MP, peak time = 90 min, FSR = 8.34%/day; CA, peak time = 120 min, FSR = 7.85%/day). Milk-derived proteins caused significantly greater increases (p protein synthesis to occur at different times (WP, fast; MP, intermediate; CA, slow) and the dairy proteins have a superior effect on muscle protein synthesis after exercise compared with SP.

  14. Predictors of muscle protein synthesis after severe pediatric burns.

    Science.gov (United States)

    Diaz, Eva C; Herndon, David N; Lee, Jinhyung; Porter, Craig; Cotter, Matthew; Suman, Oscar E; Sidossis, Labros S; Børsheim, Elisabet

    2015-04-01

    Following a major burn, skeletal muscle protein synthesis rate increases but is often insufficient to compensate for massively elevated muscle protein breakdown rates. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that muscle protein synthesis rate would be chronically elevated in severely burned children. The objectives of this study were to characterize muscle protein synthesis rate of burned children over a period of 24 months after injury and to identify predictors that influence this response. A total of 87 children with 40% or greater total body surface area (TBSA) burned were included. Patients participated in stable isotope infusion studies at 1, 2, and approximately 4 weeks after burn and at 6, 12, and 24 months after injury to determine skeletal muscle protein fractional synthesis rate. Generalized estimating equations with log link normal distribution were applied to account for clustering of patients and control for patient characteristics. Patients (8 ± 6 years) had large (62, 51-72% TBSA) and deep (47% ± 21% TBSA third degree) burns. Muscle protein fractional synthesis rate was elevated throughout the first 12 months after burn compared with established values from healthy young adults. Muscle protein fractional synthesis rate was lower in boys, in children older than 3 years, and when burns were greater than 80% TBSA. Muscle protein synthesis is elevated for at least 1 year after injury, suggesting that greater muscle protein turnover is a component of the long-term pathophysiologic response to burn trauma. Muscle protein synthesis is highly affected by sex, age, and burn size in severely burned children. These findings may explain the divergence in net protein balance and lean body mass in different populations of burn patients. Prognostic study, level III.

  15. The Networks of Genes Encoding Palmitoylated Proteins in Axonal and Synaptic Compartments Are Affected in PPT1 Overexpressing Neuronal-Like Cells

    Directory of Open Access Journals (Sweden)

    Francesco Pezzini

    2017-08-01

    Full Text Available CLN1 disease (OMIM #256730 is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1 is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1 and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2 positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs in SH-p.wtCLN1 (as compared to empty-vector transfected cells, whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45 was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to

  16. Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. I. Nuclear-directed protein synthesis.

    Science.gov (United States)

    Heude, M; Chanet, R; Moustacchi, E

    1975-04-01

    The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phage haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the "petite" recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of "petites" in stationary phase cells (increase of the frequency of "petites" during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.

  17. Protein O-Mannosyltransferases Affect Sensory Axon Wiring and Dynamic Chirality of Body Posture in the Drosophila Embryo.

    Science.gov (United States)

    Baker, Ryan; Nakamura, Naosuke; Chandel, Ishita; Howell, Brooke; Lyalin, Dmitry; Panin, Vladislav M

    2018-02-14

    Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies. POMT genes are evolutionarily conserved in metazoan organisms. In Drosophila , both male and female POMT mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in POMT mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in Drosophila development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that POMT mutants show abnormal axonal connections of sensory neurons. POMT transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in POMT mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of POMT genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies. SIGNIFICANCE STATEMENT Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in POMTs cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that POMT mutations in Drosophila result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique POMT -dependent

  18. Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron.

    Science.gov (United States)

    Locatelli, Denise; d'Errico, Paolo; Capra, Silvia; Finardi, Adele; Colciaghi, Francesca; Setola, Veronica; Terao, Mineko; Garattini, Enrico; Battaglia, Giorgio

    2012-05-01

    The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  19. Target-Derived Neurotrophins Coordinate Transcription and Transport of Bclw to Prevent Axonal Degeneration

    Science.gov (United States)

    Cosker, Katharina E.; Pazyra-Murphy, Maria F.; Fenstermacher, Sara J.

    2013-01-01

    Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit. PMID:23516285

  20. Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor.

    Science.gov (United States)

    Ma, Yihe; Bayguinov, Peter O; Jackson, Meyer B

    2017-01-01

    The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.

  1. Oligodendrocyte Development in the Absence of Their Target Axons In Vivo.

    Directory of Open Access Journals (Sweden)

    Rafael Almeida

    Full Text Available Oligodendrocytes form myelin around axons of the central nervous system, enabling saltatory conduction. Recent work has established that axons can regulate certain aspects of oligodendrocyte development and myelination, yet remarkably oligodendrocytes in culture retain the ability to differentiate in the absence of axons and elaborate myelin sheaths around synthetic axon-like substrates. It remains unclear the extent to which the life-course of oligodendrocytes requires the presence of, or signals derived from axons in vivo. In particular, it is unclear whether the specific axons fated for myelination regulate the oligodendrocyte population in a living organism, and if so, which precise steps of oligodendrocyte-cell lineage progression are regulated by target axons. Here, we use live-imaging of zebrafish larvae carrying transgenic reporters that label oligodendrocyte-lineage cells to investigate which aspects of oligodendrocyte development, from specification to differentiation, are affected when we manipulate the target axonal environment. To drastically reduce the number of axons targeted for myelination, we use a previously identified kinesin-binding protein (kbp mutant, in which the first myelinated axons in the spinal cord, reticulospinal axons, do not fully grow in length, creating a region in the posterior spinal cord where most initial targets for myelination are absent. We find that a 73% reduction of reticulospinal axon surface in the posterior spinal cord of kbp mutants results in a 27% reduction in the number of oligodendrocytes. By time-lapse analysis of transgenic OPC reporters, we find that the reduction in oligodendrocyte number is explained by a reduction in OPC proliferation and survival. Interestingly, OPC specification and migration are unaltered in the near absence of normal axonal targets. Finally, we find that timely differentiation of OPCs into oligodendrocytes does not depend at all on the presence of target axons

  2. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

    Science.gov (United States)

    Duncan, Jason E.; Lytle, Nikki K.; Zuniga, Alfredo; Goldstein, Lawrence S. B.

    2013-01-01

    Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport. PMID:23840848

  3. Slow Muscle Precursors Lay Down a Collagen XV Matrix Fingerprint to Guide Motor Axon Navigation.

    Science.gov (United States)

    Guillon, Emilie; Bretaud, Sandrine; Ruggiero, Florence

    2016-03-02

    The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development. In addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an

  4. Functional recovery of regenerating motor axons is delayed in mice heterozygously deficient for the myelin protein P(0) gene

    DEFF Research Database (Denmark)

    Rosberg, Mette Romer; Alvarez, Susana; Krarup, Christian

    2013-01-01

    Mice with a heterozygous knock-out of the myelin protein P0 gene (P0+/-) develop a neuropathy similar to human Charcot-Marie-Tooth disease. They are indistinguishable from wild-types (WT) at birth and develop a slowly progressing demyelinating neuropathy. The aim of this study was to investigate...... whether the regeneration capacity of early symptomatic P0+/- is impaired as compared to age matched WT. Right sciatic nerves were lesioned at the thigh in 7-8 months old mice. Tibial motor axons at ankle were investigated by conventional motor conduction studies and axon excitability studies using...... threshold tracking. To evaluate regeneration we monitored the recovery of motor function after crush, and then compared the fiber distribution by histology. The overall motor performance was investigated using Rotor-Rod. P0+/- had reduced compound motor action potential amplitudes and thinner myelinated...

  5. Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila

    Directory of Open Access Journals (Sweden)

    Yasmina Talmat-Amar

    2018-03-01

    Full Text Available Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs. Here, we took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous Tau functions. We found that Tau depletion leads to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, we show that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau.

  6. Axonal loss in the multiple sclerosis spinal cord revisited.

    Science.gov (United States)

    Petrova, Natalia; Carassiti, Daniele; Altmann, Daniel R; Baker, David; Schmierer, Klaus

    2018-05-01

    Preventing chronic disease deterioration is an unmet need in people with multiple sclerosis, where axonal loss is considered a key substrate of disability. Clinically, chronic multiple sclerosis often presents as progressive myelopathy. Spinal cord cross-sectional area (CSA) assessed using MRI predicts increasing disability and has, by inference, been proposed as an indirect index of axonal degeneration. However, the association between CSA and axonal loss, and their correlation with demyelination, have never been systematically investigated using human post mortem tissue. We extensively sampled spinal cords of seven women and six men with multiple sclerosis (mean disease duration= 29 years) and five healthy controls to quantify axonal density and its association with demyelination and CSA. 396 tissue blocks were embedded in paraffin and immuno-stained for myelin basic protein and phosphorylated neurofilaments. Measurements included total CSA, areas of (i) lateral cortico-spinal tracts, (ii) gray matter, (iii) white matter, (iv) demyelination, and the number of axons within the lateral cortico-spinal tracts. Linear mixed models were used to analyze relationships. In multiple sclerosis CSA reduction at cervical, thoracic and lumbar levels ranged between 19 and 24% with white (19-24%) and gray (17-21%) matter atrophy contributing equally across levels. Axonal density in multiple sclerosis was lower by 57-62% across all levels and affected all fibers regardless of diameter. Demyelination affected 24-48% of the gray matter, most extensively at the thoracic level, and 11-13% of the white matter, with no significant differences across levels. Disease duration was associated with reduced axonal density, however not with any area index. Significant association was detected between focal demyelination and decreased axonal density. In conclusion, over nearly 30 years multiple sclerosis reduces axonal density by 60% throughout the spinal cord. Spinal cord cross sectional area

  7. Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

    Directory of Open Access Journals (Sweden)

    John P Cavaretta

    Full Text Available KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4 membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal

  8. The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

    Science.gov (United States)

    Mizutani, H.; Ponnamperuma, C.

    1977-01-01

    A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

  9. Liver protein synthesis stays elevated after chemotherapy in tumour-bearing mice.

    Science.gov (United States)

    Samuels, Sue E; McLaren, Teresa A; Knowles, Andrew L; Stewart, Sarah A; Madelmont, Jean-Claude; Attaix, Didier

    2006-07-28

    We studied the effect of chemotherapy on liver protein synthesis in mice bearing colon 26 adenocarcinoma (C26). Liver protein mass decreased (-32%; Psynthesis increased (20-35%; Psynthesis. Increased protein synthesis in tumour-bearing mice was primarily mediated by increasing ( approximately 15%; Psynthesis (Cs; mg RNA/g protein). Cystemustine, a nitrosourea chemotherapy that cures C26 with 100% efficacy, rapidly restored liver protein mass; protein synthesis however stayed higher than in healthy mice ( approximately 15%) throughout the initial and later stages of recovery. Chemotherapy had no significant effect on liver protein mass and synthesis in healthy mice. Reduced food intake was not a factor in this model. These data suggest a high priority for liver protein synthesis during cancer cachexia and recovery.

  10. Age-related changes in the synthesis and phosphorylation of proteins

    International Nuclear Information System (INIS)

    Butler, J.A.; Heydari, A.; Richardson, A.

    1986-01-01

    It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either [ 3 H]-valine or [ 32 P]-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age

  11. Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

    Science.gov (United States)

    Spitzbarth, Ingo; Lempp, Charlotte; Kegler, Kristel; Ulrich, Reiner; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Seehusen, Frauke

    2016-07-01

    CDV-DL (Canine distemper virus-induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination. Immunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed. Increased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up-regulated in the chronic stage compared to dogs with subacute CDV-DL. However, immunohistochemically, hints for axonal regeneration were restricted to up-regulated axonal positivity of hypoxia-inducible factor 1 alpha, while growth-associated protein 43, erythropoietin and its receptor were not or even down-regulated. Periaxin-positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions. The present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV-DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

  12. Retinal protein synthesis in relationship to environmental lighting

    International Nuclear Information System (INIS)

    Hollyfield, J.G.; Anderson, R.E.

    1982-01-01

    A series of in vivo and in vitro experiments using Xenopus laevis juvenile toads was conducted to probe the relationship between environmental lighting and protein synthesis in the retina. Autoradiographic and biochemical analyses indicated that measurable changes in protein synthesis did not occur during a normal diurnal cycle when animals were conditioned to 12 hr light followed by 12 hr darkness each day (LD). However, when retinas from animals maintained in continuous darkness (DD) for 3 days were incubated with 3 H-leucine, there was a 40% reduction in the specific radioactivity of total retinal proteins compared with retinas from animals maintained in continuous light (LL) for 3 days or on the LD cycle. Retinas from DD animals injected with 3 H-leucine showed a 48% reduction in protein synthesis compared with retinas of LL animals. In autoradiographs of retinas from in vivo or in vitro experiments, grain counts were 40% lower in the total retinas of the DD animals compared with retinas of LL animals. This reduction occurred throughout the entire retina and was not restricted to any specific cell type. There was also a 35% reduction in the rate of radioactive band displacement in the rod outer segments of DD animals, although the percent of 3 H-leucine incorporated into opsin relative to total retinal protein was the same for both groups. We conclude from these studies that fluctuations in the rate of protein synthesis during the normal light-dark cycle are not detectable. However, major differences in protein synthesis are evident when animals are stressed with continuous darkness for several days. This effect is not restricted to any particular retinal layer but occurs throughout the entire retina. Moreover, prolonged darkness affects protein synthesis in extraocular tissues as well

  13. A low-protein diet restricts albumin synthesis in nephrotic rats.

    OpenAIRE

    Kaysen, G A; Jones, H; Martin, V; Hutchison, F N

    1989-01-01

    High-protein diets increase albumin synthesis in rats with Heymann nephritis but albuminuria increases also, causing serum albumin concentration to be suppressed further than in nephrotic animals eating a low-protein diet. Experiments were designed to determine whether dietary protein augmentation directly stimulates albumin synthesis, or whether instead increased albumin synthesis is triggered by the decrease in serum albumin concentration. Evidence is presented that dietary protein augmenta...

  14. Inhibition of chloroplast protein synthesis following light chilling of tomato

    International Nuclear Information System (INIS)

    Kent, J.; Ort, D.

    1989-01-01

    In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

  15. The genetics of axonal transport and axonal transport disorders.

    Directory of Open Access Journals (Sweden)

    Jason E Duncan

    2006-09-01

    Full Text Available Neurons are specialized cells with a complex architecture that includes elaborate dendritic branches and a long, narrow axon that extends from the cell body to the synaptic terminal. The organized transport of essential biological materials throughout the neuron is required to support its growth, function, and viability. In this review, we focus on insights that have emerged from the genetic analysis of long-distance axonal transport between the cell body and the synaptic terminal. We also discuss recent genetic evidence that supports the hypothesis that disruptions in axonal transport may cause or dramatically contribute to neurodegenerative diseases.

  16. Optimizing the measurement of mitochondrial protein synthesis in human skeletal muscle.

    Science.gov (United States)

    Burd, Nicholas A; Tardif, Nicolas; Rooyackers, Olav; van Loon, Luc J C

    2015-01-01

    The measurement of mitochondrial protein synthesis after food ingestion, contractile activity, and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the longer term. Studies have shown that protein ingestion stimulates mitochondrial protein synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be no measurable differences in mitochondrial protein synthesis between critically ill patients and aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a more advanced age. In this paper, we discuss the challenges involved in the measurement of human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid tracer methods. Practical guidelines are discussed to improve the reliability of the measurement of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle mass and performance outcomes in both the healthy and disease populations requires more work, but we emphasize that the measurements need to be reliable to be of any value to the field.

  17. Protein synthesis in the presence of carbamoyl-amino acids

    International Nuclear Information System (INIS)

    Kraus, L.M.; Stephens, M.C.

    1987-01-01

    The role of exogenous carbamoyl-amino acids in protein biosynthesis has been examined in vitro using a mixture of 14 C amino acids to label newly synthesized protein in human reticulocyte rich (8-18%) peripheral blood. Aliquots of the radiolabeled newly synthesized protein were acid precipitated, washed and the radioactivity measured. Control samples which measured the synthetic capacity of the blood were aliquots of the same blood- 14 C amino acid mixture without added carbamoyl-amino acids or cyanate. N-carbamoyl leucine alone or a 3 N-carbamoyl amino acid mixture of leucine, aspartic acid and tyrosine were used to test inhibition of protein synthesis. Also carbamoyl-amino acids were synthesized using cyanate and Pierce hydrolyzate amino acid calibration standards or the mixture of 14 C amino acids. In this system the carbamoylation of endogenous amino acids by cyanate up to 8 μmol/100μl showed a linear decrease in protein synthesis with time which is inversely related to the cyanate concentration. At greater cyanate levels the inhibition of protein synthesis reaches a plateau. When N-carbamoyl-amino acids only are present there is about a 50% decrease in the 14 C protein at 30 minutes as compared to the synthesis of 14 C protein without N-carbamoyl-amino acids. These results indicate that the presence of carbamoyl-amino acids interferes with protein synthesis

  18. 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.

  19. Subtypes of GABAergic neurons project axons in the neocortex

    Directory of Open Access Journals (Sweden)

    Shigeyoshi Higo

    2009-11-01

    Full Text Available γ-aminobutyric acid (GABAergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP-labeled GABAergic neurons in GAD67-Cre knock-in / GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral cortex and are in a position to be involved in information processing.

  20. Impaired Mitochondrial Dynamics Underlie Axonal Defects in Hereditary Spastic Paraplegias.

    Science.gov (United States)

    Denton, Kyle; Mou, Yongchao; Xu, Chong-Chong; Shah, Dhruvi; Chang, Jaerak; Blackstone, Craig; Li, Xue-Jun

    2018-05-02

    Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

  1. Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase

    International Nuclear Information System (INIS)

    Olsen, W.A.; Perchellet, E.; Malinowski, R.L.

    1986-01-01

    The effects of insulin deficiency on nitrogen metabolism in muscle and liver have been extensively studied with recent in vivo demonstration of impaired protein synthesis in rats with streptozotocin-induced diabetes. Despite the significant contribution of small intestinal mucosa to overall protein metabolism, the effect of insulin deficiency on intestinal protein synthesis have not been completely defined. The authors studied the effects of streptozotocin-induced diabetes on total protein synthesis by small intestinal mucosa and on synthesis of a single enzyme protein of the enterocyte brush-border membrane sucrase-isomaltase. They used the flood-dose technique to minimize the difficulties of measuring specific radioactivity of precursor phenylalanine and determined incorporation into mucosal proteins and sucrase-isomaltase 20 min after injection of the labeled amino acid. Diabetes did not alter mucosal mass as determined by weight and content of protein and DNA during the 5 days after injection of streptozotocin. Increased rates of sucrase-isomaltase synthesis developed beginning on day 3, and those of total protein developed on day 5. Thus intestinal mucosal protein synthesis is not an insulin-sensitive process

  2. Constitutively expressed Protocadherin-α regulates the coalescence and elimination of homotypic olfactory axons through its cytoplasmic region

    Directory of Open Access Journals (Sweden)

    Sonoko eHasegawa

    2012-10-01

    Full Text Available Olfactory sensory neuron (OSN axons coalesce into specific glomeruli in the olfactory bulb (OB according to their odorant receptor (OR expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α’s cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

  3. Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

    Science.gov (United States)

    Garrity, P A; Rao, Y; Salecker, I; McGlade, J; Pawson, T; Zipursky, S L

    1996-05-31

    Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

  4. IFNgamma enhances microglial reactions to hippocampal axonal degeneration

    DEFF Research Database (Denmark)

    Jensen, M B; Hegelund, I V; Lomholt, N D

    2000-01-01

    periods. Message for the immune cytokine interferon-gamma (IFNgamma) was undetectable, and glial reactivity to axonal lesions occurred as normal in IFNgamma-deficient mice. Microglial responses to lesion-induced neuronal injury were markedly enhanced in myelin basic protein promoter-driven transgenic mice...

  5. Regulation of protein synthesis during sea urchin early development

    International Nuclear Information System (INIS)

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

  6. Adeno-associated virus rep protein synthesis during productive infection

    International Nuclear Information System (INIS)

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-01-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [ 35 S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased

  7. Dendritic protein synthesis in the normal and diseased brain

    Science.gov (United States)

    Swanger, Sharon A.; Bassell, Gary J.

    2015-01-01

    Synaptic activity is a spatially-limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases. PMID:23262237

  8. Acetylated tau destabilizes the cytoskeleton in the axon initial segment and is mislocalized to the somatodendritic compartment.

    Science.gov (United States)

    Sohn, Peter Dongmin; Tracy, Tara E; Son, Hye-In; Zhou, Yungui; Leite, Renata E P; Miller, Bruce L; Seeley, William W; Grinberg, Lea T; Gan, Li

    2016-06-29

    Neurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer's disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood. Here we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and βIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures. Together, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS.

  9. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    Science.gov (United States)

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  10. Understanding Protein Synthesis: An Interactive Card Game Discussion

    Science.gov (United States)

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a…

  11. Acute myotube protein synthesis regulation by IL-6-related cytokines.

    Science.gov (United States)

    Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

    2017-11-01

    IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the

  12. MuSC is involved in regulating axonal fasciculation of mouse primary vestibular afferents.

    Science.gov (United States)

    Kawauchi, Daisuke; Kobayashi, Hiroaki; Sekine-Aizawa, Yoko; Fujita, Shinobu C; Murakami, Fujio

    2003-10-01

    Regulation of axonal fasciculation plays an important role in the precise patterning of neural circuits. Selective fasciculation contributes to the sorting of different types of axons and prevents the misrouting of axons. However, axons must defasciculate once they reach the target area. To study the regulation of fasciculation, we focused on the primary vestibulo-cerebellar afferents (PVAs), which show a dramatic change from fasciculated axon bundles to defasciculated individual axons at their target region, the cerebellar primordium. To understand how fasciculation and defasciculation are regulated in this system, we investigated the roles of murine SC1-related protein (MuSC), a molecule belonging to the immunoglobulin superfamily. We show: (i) by comparing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) labelling and anti-MuSC immunohistochemistry, that downregulation of MuSC in PVAs during development is concomitant with the defasciculation of PVA axons; (ii) in a binding assay with cells expressing MuSC, that MuSC has cell-adhesive activity via a homophilic binding mechanism, and this activity is increased by multimerization; and (iii) that MuSC also displays neurite outgrowth-promoting activity in vestibular ganglion cultures. These findings suggest that MuSC is involved in axonal fasciculation and its downregulation may help to initiate the defasciculation of PVAs.

  13. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    OpenAIRE

    Leventhal, J M; Chambliss, G H

    1982-01-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phos...

  14. Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice

    Directory of Open Access Journals (Sweden)

    Coralie Fassier

    2013-01-01

    Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP, a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

  15. The origin of polynucleotide-directed protein synthesis

    Science.gov (United States)

    Orgel, Leslie E.

    1989-01-01

    If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

  16. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  17. Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

    Science.gov (United States)

    Tucker, K. R.; Seider, M. J.; Booth, F. W.

    1981-01-01

    Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

  18. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    International Nuclear Information System (INIS)

    Minor, P.D.; Dimmock, N.J.

    1977-01-01

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  19. Roles of Fe-S proteins: from cofactor synthesis to iron homeostasis to protein synthesis.

    Science.gov (United States)

    Pain, Debkumar; Dancis, Andrew

    2016-06-01

    Fe-S cluster assembly is an essential process for all cells. Impairment of Fe-S cluster assembly creates diseases in diverse and surprising ways. In one scenario, the loss of function of lipoic acid synthase, an enzyme with Fe-S cluster cofactor in mitochondria, impairs activity of various lipoamide-dependent enzymes with drastic consequences for metabolism. In a second scenario, the heme biosynthetic pathway in red cell precursors is specifically targeted, and iron homeostasis is perturbed, but lipoic acid synthesis is unaffected. In a third scenario, tRNA modifications arising from action of the cysteine desulfurase and/or Fe-S cluster proteins are lost, which may lead to impaired protein synthesis. These defects can then result in cancer, neurologic dysfunction or type 2 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

    Directory of Open Access Journals (Sweden)

    Boyce Mark

    2012-08-01

    Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

  1. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

    Science.gov (United States)

    Boyce, Mark; Celma, Cristina C P; Roy, Polly

    2012-08-29

    Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

  2. Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

    Science.gov (United States)

    Fallini, Claudia; Donlin-Asp, Paul G; Rouanet, Jeremy P; Bassell, Gary J; Rossoll, Wilfried

    2016-03-30

    Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization

  3. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, S.S.; Lyerly, D.P. (Environmental Protection Agency, Research Triangle Park, NC (USA))

    1989-12-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with (35S)methionine and (3H)fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure.

  4. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    International Nuclear Information System (INIS)

    Padilla, S.S.; Lyerly, D.P.

    1989-01-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with [35S]methionine and [3H]fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure

  5. Injury-induced inhibition of small intestinal protein and nucleic acid synthesis

    International Nuclear Information System (INIS)

    Carter, E.A.; Hatz, R.A.; Yarmush, M.L.; Tompkins, R.G.

    1990-01-01

    Small intestinal mucosal weight and nutrient absorption are significantly diminished early after cutaneous thermal injuries. Because these intestinal properties are highly dependent on rates of nucleic acid and protein synthesis, in vivo incorporation of thymidine, uridine, and leucine into small intestinal deoxyribonucleic acid, ribonucleic acid, and proteins were measured. Deoxyribonucleic acid synthesis was markedly decreased with the lowest thymidine incorporation in the jejunum (p less than 0.01); these findings were confirmed by autoradiographic identification of radiolabeled nuclei in the intestinal crypts. Protein synthesis was decreased by 6 h postinjury (p less than 0.01) but had returned to normal by 48 h. Consistent with a decreased rate of protein synthesis, ribonucleic acid synthesis was also decreased 18 h postinjury (p less than 0.01). These decreased deoxyribonucleic acid, ribonucleic acid, and protein synthesis rates are not likely a result of ischemia because in other studies of this injury model, intestinal blood flow was not significantly changed by the burn injury. Potentially, factors initiating the acute inflammatory reaction may directly inhibit nucleic acid and protein synthesis and lead to alterations in nutrient absorption and intestinal barrier function after injury

  6. The Influence of Glutamate on Axonal Compound Action Potential In Vitro.

    Science.gov (United States)

    Abouelela, Ahmed; Wieraszko, Andrzej

    2016-01-01

    Background  Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that glutamate released from axons could be involved in this phenomenon, the modification of the axonal CAP induced by glutamate was investigated. Objectives  The major objective of this research is to verify the hypothesis that axonal activity would trigger the release of glutamate, which in turn would interact with specific axonal receptors modifying the amplitude of the action potential. Methods  Segments of the sciatic nerve were exposed to exogenous glutamate in vitro, and CAP was recorded before and after glutamate application. In some experiments, the release of radioactive glutamate analog from the sciatic nerve exposed to exogenous glutamate was also evaluated. Results  The glutamate-induced increase in CAP was blocked by different glutamate receptor antagonists. The effect of glutamate was not observed in Ca-free medium, and was blocked by antagonists of calcium channels. Exogenous glutamate, applied to the segments of sciatic nerve, induced the release of radioactive glutamate analog, demonstrating glutamate-induced glutamate release. Immunohistochemical examination revealed that axolemma contains components necessary for glutamatergic neurotransmission. Conclusion  The proteins of the axonal membrane can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance, leading to a change in the amplitude of CAP. We suggest that increased axonal activity leads to the release of glutamate that results in changes in the amplitude of CAPs.

  7. Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis

    DEFF Research Database (Denmark)

    Doessing, Simon; Heinemeier, Katja M; Holm, Lars

    2010-01-01

    young individuals. rhGH administration caused an increase in serum GH, serum IGF-I, and IGF-I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9-fold and 1.3-fold, respectively (P ...RNA expression and muscle collagen protein synthesis increased by 2.3-fold and 5.8-fold, respectively (P protein synthesis was unaffected by elevation of GH and IGF-I. Moderate exercise did not enhance the effects of GH manipulation. Thus, increased GH availability stimulates...... matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue....

  8. Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis.

    Science.gov (United States)

    Liu, Han-Hsuan; Cline, Hollis T

    2016-07-06

    Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning

  9. Blast overpressure induced axonal injury changes in rat brainstem and spinal cord

    Directory of Open Access Journals (Sweden)

    Srinivasu Kallakuri

    2015-01-01

    Full Text Available Introduction: Blast induced neurotrauma has been the signature wound in returning soldiers from the ongoing wars in Iraq and Afghanistan. Of importance is understanding the pathomechansim(s of blast overpressure (OP induced axonal injury. Although several recent animal models of blast injury indicate the neuronal and axonal injury in various brain regions, animal studies related to axonal injury in the white matter (WM tracts of cervical spinal cord are limited. Objective: The purpose of this study was to assess the extent of axonal injury in WM tracts of cervical spinal cord in male Sprague Dawley rats subjected to a single insult of blast OP. Materials and Methods: Sagittal brainstem sections and horizontal cervical spinal cord sections from blast and sham animals were stained by neurofilament light (NF-L chain and beta amyloid precursor protein immunocytochemistry and observed for axonal injury changes. Results: Observations from this preliminary study demonstrate axonal injury changes in the form of prominent swellings, retraction bulbs, and putative signs of membrane disruptions in the brainstem and cervical spinal cord WM tracts of rats subjected to blast OP. Conclusions: Prominent axonal injury changes following the blast OP exposure in brainstem and cervical spinal WM tracts underscores the need for careful evaluation of blast induced injury changes and associated symptoms. NF-L immunocytochemistry can be considered as an additional tool to assess the blast OP induced axonal injury.

  10. Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

    OpenAIRE

    sprotocols

    2015-01-01

    Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

  11. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    Science.gov (United States)

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

  12. Synthesis of stress proteins in winter wheat seedlings under gamma-radiation

    International Nuclear Information System (INIS)

    Gudkova, N.V.; Kosakovskaya, I.V.; Major, P.S.

    2001-01-01

    A universal cellular response to a number of diverse stresses is the synthesis of a set of stress proteins. Most of them are heat shock proteins (HSP). We show that both heat shock and gamma-radiation enhance the synthesis of HSP70 in the total protein fractions of winter wheat seedlings. It is found that a dose of 15 Gy induced the synthesis of 35 and 45 kD proteins after 5 h of irradiation in both total and mitochondrial protein fractions. On the second day after exposure, both 35 and 45 kD proteins were not observed, but new total proteins with a molecular weight of 90 and 92 kD appeared. The synthesis of 35 and 45 kD proteins after gamma-irradiation is revealed for the first time, their function being now unknown

  13. RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 2

    International Nuclear Information System (INIS)

    Skog, S.; Tribukait, B.; Nygard, O.; Wenner-Gren-Center foer Vetenskaplig Forskning, Stockholm

    1985-01-01

    Poly(A)-containing RNA (m-RNA) was studied in in vivo growing Ehrlich ascites tumour cells following a roentgen irradiation dose of 5 Gy. m-RNA increased significantly during the first 12 hours after irradiation. Thus, the observed decrease in protein synthesis rate during this time seems not to be due to radiation induced changes at the transcriptional level. The protein synthesis rate of in vivo irradiated cells incubated in vitro in culture medium was unchanged. On the other hand, the protein synthesis rate of non-irradiated cells incubated in vitro in ascites fluid from irradiated animals was decreased. We concluded that factor(s) inhibiting protein synthesis or the lack of factor(s) promoting protein synthesis in the ascites fluid is(are) of significance for the reduced protein synthesis of tumour cells found in irradiated in vivo growing cells. (orig.)

  14. Induction of neuronal axon outgrowth by Shati/Nat8l by energy metabolism in mice cultured neurons.

    Science.gov (United States)

    Sumi, Kazuyuki; Uno, Kyosuke; Matsumura, Shohei; Miyamoto, Yoshiaki; Furukawa-Hibi, Yoko; Muramatsu, Shin-Ichi; Nabeshima, Toshitaka; Nitta, Atsumi

    2015-09-09

    A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens of mice repeatedly treated with methamphetamine (METH). Shati/Nat8l has been reported to inhibit the pharmacological action induced by METH. Shati/Nat8l produces N-acetylaspartate from aspartate and acetyl-CoA. Previously, we reported that overexpression of Shati/Nat8l in nucleus accumbens attenuates the response to METH by N-acetylaspartylglutamate (which is derived from N-acetylaspartate)-mGluR3 signaling in the mice brain. In the present study, to clarify the type of cells that produce Shati/Nat8l, we carried out in-situ hybridization for the detection of Shati/Nat8l mRNA along with immunohistochemical studies using serial sections of mice brain. Shati/Nat8l mRNA was detected in neuronal cells, but not in astrocytes or microglia cells. Next, we investigated the function of Shati/Nat8l in the neuronal cells in mice brain; then, we used an adeno-associated virus vector containing Shati/Nat8l for transfection and overexpression of Shati/Nat8l protein into the primary cultured neurons to investigate the contribution toward the neuronal activity of Shati/Nat8l. Overexpression of Shati/Nat8l in the mice primary cultured neurons induced axonal growth, but not dendrite elongation at day 1.5 (DIV). This finding indicated that Shati/Nat8l contributes toward neuronal development. LY341495, a selective group II mGluRs antagonist, did not abolish this axonal growth, and N-acetylaspartylglutamate itself did not abolish axon outgrowth in the same cultured system. The cultured neurons overexpressing Shati/Nat8l contained high ATP, suggesting that axon outgrowth is dependent on energy metabolism. This study shows that Shati/Nat8l in the neuron may induce axon outgrowth by ATP synthesis and not through mGluR3 signaling.

  15. Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

    Directory of Open Access Journals (Sweden)

    Yihao Zhang

    2017-02-01

    Full Text Available Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav, which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

  16. Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

    Science.gov (United States)

    Zhang, Yihao; Abiraman, Krithika; Li, He; Pierce, David M; Tzingounis, Anastasios V; Lykotrafitis, George

    2017-02-01

    Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

  17. Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits

    International Nuclear Information System (INIS)

    Samarel, A.M.; Parmacek, M.S.; Magid, N.M.; Decker, R.S.; Lesch, M.

    1987-01-01

    To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [ 3 H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting

  18. Protein synthesis and sublethal damage repair in synchronized CHO cells

    International Nuclear Information System (INIS)

    Yezzi, M.J.; Tobias, C.A.; Blakely, E.A.

    1984-01-01

    The authors have previously reported that the split dose survival response to x-rays of asynchronous CHO-TSH1 cells is reduced if the cells are held at 40 0 C,a temperature that inhibits protein synthesis, for 2 hours before the first dose and during a 2-hour interval between doses. In conjunction with the survival experiments on asynchronous cells, the authors also examined the DNA rejoining ability in split dose studies with and without inhibition of protein synthesis. The results of these experiments suggest that inhibition of protein synthesis affects a pool of proteins that are necessary for the correct expression of the DNA, although they do not appear to be involved in rejoining DNA breaks. They have extended this work to the study of cells synchronized in G1 phase (2 hour post-mitosis) and S phase (10 hour post-mitosis). Autoradiographic analyses, using 3H-TdR pulse labeling, demonstrated that a delay in the progression of each synchronized cell population occurs after inhibition of protein synthesis. Data are reported on the effects of inhibition of protein synthesis on the ability of G1 and S phase cells to repair sublethal damage

  19. Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging

    Directory of Open Access Journals (Sweden)

    Kohara Keigo

    2005-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF, which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP-tagged BDNF in axons and dendrites of living cortical neurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP was compared with that of nerve growth factor (NGF tagged with yellow fluorescent protein (YFP, to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin. Results We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s. Conclusion The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.

  20. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials

    NARCIS (Netherlands)

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; Duinkerken, Van Gert; Yu, Peiqiang

    2015-01-01

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more

  1. Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

    OpenAIRE

    Jablonka, Sibylle; Beck, Marcus; Lechner, Barbara Dorothea; Mayer, Christine; Sendtner, Michael

    2007-01-01

    Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced β-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced f...

  2. PROSPECTIVE TEACHERS’ COGNITIVE STRUCTURES CONCERNING PROTEIN SYNTHESIS AND THEIR DEGREE OF UNDERSTANDING

    Directory of Open Access Journals (Sweden)

    Cem Gerçek

    2018-02-01

    Full Text Available The purpose of education is to actualise meaningful learning. Therefore, researching the issues on how students process information and how they configure it is important for meaningful learning. The issue of protein synthesis contains a number of abstract topics and concepts. Hence, it is important in biology teaching to be informed of students’ cognitive structures concerning protein synthesis. This research aims to analyse prospective teachers’ cognitive structures about protein synthesis and their degree of understanding the subject. The research group was composed of 17 volunteering prospective teachers who had been chosen through purposeful sampling. The data were collected via semi-structured interviews. Flow maps and content analysis were used in analysing the data. The results demonstrated that prospective teachers had too many misconceptions about protein synthesis and that their knowledge extent and rich connection are inadequate. The prospective teachers’ degree of understanding protein synthesis was divided into three categories. The results obtained in this research suggested that teachers should be careful in teaching the subject of protein synthesis. Students’ prior knowledge and their misconceptions should be determined and content or contexts to facilitate them to learn an abstract subject such as protein synthesis should be presented.

  3. Response of rat brain protein synthesis to ethanol and sodium barbital

    International Nuclear Information System (INIS)

    Tewari, S.; Greenberg, S.A.; Do, K.; Grey, P.A.

    1987-01-01

    Central nervous system (CNS) depressants such as ethanol and barbiturates under acute or chronic conditions can induce changes in rat brain protein synthesis. While these data demonstrate the individual effects of drugs on protein synthesis, the response of brain protein synthesis to alcohol-drug interactions is not known. The goal of the present study was to determine the individual and combined effects of ethanol and sodium barbital on brain protein synthesis and gain an understanding of the mechanisms by which these alterations in protein synthesis are produced. Specifically, the in vivo and in vitro effects of sodium barbital (one class of barbiturates which is not metabolized by the hepatic tissue) were examined on brain protein synthesis in rats made physically dependent upon ethanol. Using cell free brain polysomal systems isolated from Control, Ethanol and 24 h Ethanol Withdrawn rats, data show that sodium barbital, when intubated intragastrically, inhibited the time dependent incorporation of 14 C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the 14 C) leucine incorporation into protein when compared to the Control and Ethanol groups. In addition, sodium barbital when added at various concentrations in vitro to the incubation medium inhibited the incorporation of 14 C) leucine into protein by Control and Ethanol polysomes. The inhibitory effects were also obtained following preincubation of ribosomes in the presence of barbital but not cycloheximide. Data suggest that brain protein synthesis, specifically brain polysomes, through interaction with ethanol or barbital are involved in the functional development of tolerance. These interactions may occur through proteins or polypeptide chains or alterations in messenger RNA components associated with the ribosomal units

  4. hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons.

    Science.gov (United States)

    Briese, Michael; Saal-Bauernschubert, Lena; Ji, Changhe; Moradi, Mehri; Ghanawi, Hanaa; Uhl, Michael; Appenzeller, Silke; Backofen, Rolf; Sendtner, Michael

    2018-03-20

    Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance. Copyright © 2018 the Author(s). Published by PNAS.

  5. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    Science.gov (United States)

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  6. Axonal transport and axon sprouting in the adult rat dentate gyrus: an autoradiographic study

    International Nuclear Information System (INIS)

    Goldowitz, D.; Cotman, C.W.

    1980-01-01

    In response to an entorhinal lesion, the commissural and associational afferents to the dentate gyrus have been shown to expand beyond their normal terminal zone into the area denervated by the entorhinal lesion. The present study has investigated the axonal transport of [ 3 H]-labeled proteins in the commissural and associational projections following an entorhinal lesion. Injections of [ 3 H]proline, [ 3 H]leucine or [ 3 H)fucose were given in the vicinity of the commissural and associational cells of origin before, immediately subsequent to, or at 5 to 15 days after the entorhinal lesion. The disposition of previously- or newly-synthesized proteins was examined in the commissural and associational terminal field at different times after an entorhinal lesion by light-microscopic autoradiography. (author)

  7. Axonal transport and axon sprouting in the adult rat dentate gyrus: an autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Goldowitz, D; Cotman, C W [California Univ., Irvine (USA)

    1980-12-01

    In response to an entorhinal lesion, the commissural and associational afferents to the dentate gyrus have been shown to expand beyond their normal terminal zone into the area denervated by the entorhinal lesion. The present study has investigated the axonal transport of (/sup 3/H)-labeled proteins in the commissural and associational projections following an entorhinal lesion. Injections of (/sup 3/H)proline, (/sup 3/H)leucine or (/sup 3/H)fucose were given in the vicinity of the commissural and associational cells of origin before, immediately subsequent to, or at 5 to 15 days after the entorhinal lesion. The disposition of previously- or newly-synthesized proteins was examined in the commissural and associational terminal field at different times after an entorhinal lesion by light-microscopic autoradiography.

  8. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis.

    Science.gov (United States)

    Winkle, Cortney C; Hanlin, Christopher C; Gupton, Stephanie L

    2016-03-16

    During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

  9. Signal propagation along the axon.

    Science.gov (United States)

    Rama, Sylvain; Zbili, Mickaël; Debanne, Dominique

    2018-03-08

    Axons link distant brain regions and are usually considered as simple transmission cables in which reliable propagation occurs once an action potential has been generated. Safe propagation of action potentials relies on specific ion channel expression at strategic points of the axon such as nodes of Ranvier or axonal branch points. However, while action potentials are generally considered as the quantum of neuronal information, their signaling is not entirely digital. In fact, both their shape and their conduction speed have been shown to be modulated by activity, leading to regulations of synaptic latency and synaptic strength. We report here newly identified mechanisms of (1) safe spike propagation along the axon, (2) compartmentalization of action potential shape in the axon, (3) analog modulation of spike-evoked synaptic transmission and (4) alteration in conduction time after persistent regulation of axon morphology in central neurons. We discuss the contribution of these regulations in information processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

    Lawton, K.A.; Raghothama, K.G.; Woodson, W.R.

    1990-01-01

    We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

  11. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    International Nuclear Information System (INIS)

    Sampson, D.A.; Jansen, G.R.

    1985-01-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of [3- 3 H]phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis

  12. Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

    Science.gov (United States)

    Kent, Stephen Bh

    2018-04-04

    A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Ethylene and protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Osborne, D J

    1973-01-01

    Ethylene reduces the rate of expansion growth of cells and it is suggestive that the rate of expansion is controlled at least in part by the synthesis of hydroxyproline rich glycopeptides that are secreted with other polysaccharide material through the plasmalemma into the cell wall, thereby enhancing the thickness of the cell wall and also rendering it poorly extensible. In combination, auxin would appear to counteract the effect of ethylene in this respect, for although auxin enhances the synthesis of protein and the content in the cell walls, as well as causing some increase in wall thickness, it reduces the amount of hydroxyproline reaching the wall. Such effects may be instrumental in enhancing wall plasticity, the rate of expansion and the final cell size. These results indicate that ethylene and auxin together afford a dual regulatory system exerted through a control of a specific part of the protein synthetic pathway, the products of which regulate the rate of expansion, and the potential for expansion, of the plant cell wall. 38 references, 3 figures, 8 tables.

  14. Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work.

    Science.gov (United States)

    Kent, Stephen

    2017-09-15

    Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation - thioester-mediated, amide-forming reaction at Xaa-Cys sites - and its extensions. Case studies from the author's own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Paired Immunoglobulin-like Receptor B Knockout Does Not Enhance Axonal Regeneration or Locomotor Recovery after Spinal Cord Injury*

    OpenAIRE

    Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

    2010-01-01

    Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tr...

  16. Cortical compression rapidly trimmed transcallosal projections and altered axonal anterograde transport machinery.

    Science.gov (United States)

    Chen, Li-Jin; Wang, Yueh-Jan; Tseng, Guo-Fang

    2017-10-24

    Trauma and tumor compressing the brain distort underlying cortical neurons. Compressed cortical neurons remodel their dendrites instantly. The effects on axons however remain unclear. Using a rat epidural bead implantation model, we studied the effects of unilateral somatosensory cortical compression on its transcallosal projection and the reversibility of the changes following decompression. Compression reduced the density, branching profuseness and boutons of the projection axons in the contralateral homotopic cortex 1week and 1month post-compression. Projection fiber density was higher 1-month than 1-week post-compression, suggesting adaptive temporal changes. Compression reduced contralateral cortical synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and postsynaptic density protein-95 (PSD95) expressions in a week and the first two marker proteins further by 1month. βIII-tubulin and kinesin light chain (KLC) expressions in the corpus callosum (CC) where transcallosal axons traveled were also decreased. Kinesin heavy chain (KHC) level in CC was temporarily increased 1week after compression. Decompression increased transcallosal axon density and branching profuseness to higher than sham while bouton density returned to sham levels. This was accompanied by restoration of synaptophysin, VGLUT1 and PSD95 expressions in the contralateral cortex of the 1-week, but not the 1-month, compression rats. Decompression restored βIII-tubulin, but not KLC and KHC expressions in CC. However, KLC and KHC expressions in the cell bodies of the layer II/III pyramidal neurons partially recovered. Our results show cerebral compression compromised cortical axonal outputs and reduced transcallosal projection. Some of these changes did not recover in long-term decompression. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Axons take a dive

    Science.gov (United States)

    Tong, Cheuk Ka; Cebrián-Silla, Arantxa; Paredes, Mercedes F; Huang, Eric J; García-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2015-01-01

    In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular–subventricular zone (V–SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells. PMID:26413556

  18. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

  19. Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

    Directory of Open Access Journals (Sweden)

    Araceli eMartínez-Moreno

    2011-09-01

    Full Text Available It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memory when protein synthesis was inhibited. Our previous studies on the insular cortex (IC, a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA, have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis dependent in different time-windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 hours after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  20. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement.

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F; Escobar, Martha L

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  1. Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

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    Richa Tyagi

    2015-02-01

    Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

  2. Rewiring protein synthesis: From natural to synthetic amino acids.

    Science.gov (United States)

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2017-11-01

    The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Axonal Spheroid Accumulation In the Brainstem and Spinal Cord of A Young Angus Cow with Ataxia.

    Science.gov (United States)

    Hanshaw, D M; Finnie, J W; Manavis, J; Kessell, A E

    2015-08-01

    An 18-month-old Angus cow presented with rapidly developing ataxia and subsequently died. The finding of large numbers of axonal spheroids in brainstem nuclei and spinal cord grey matter, bilaterally symmetrical in distribution, was consistent with a histopathological diagnosis of neuroaxonal dystrophy (NAD). Most of the axonal swellings were immunopositive to amyloid precursor protein, suggesting that interruption to axonal flow was important in their genesis. The topographical distribution of axonal spheroids in the brain and spinal cord in this bovine case closely resembled that found in the ovine neurodegenerative disorder termed NAD, in which axonal swellings are the major pathological feature. This appears to be the first reported case of this type of NAD in cattle. The aetiology of the spheroidal aggregations in this case was not determined. There was no evidence from the case history or neuropathology to indicate whether the axonal spheroids in this case involved an acquired or heritable aetiology. © 2015 Australian Veterinary Association.

  4. Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

    Science.gov (United States)

    Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

    2017-01-01

    Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older men after a day of bed rest, the application of NMES prior to presleep protein feeding stimulates the use of

  5. Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

    Science.gov (United States)

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

    2016-09-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P protein synthesis rates increased after ingesting MCas-35 (P protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an equal amount of wheat protein. Ingesting a larger amount of wheat protein (i.e., 60 g) substantially increases myofibrillar protein synthesis rates in healthy older men. This trial was registered at clinicaltrials.gov as NCT01952639. © 2016 American Society for Nutrition.

  6. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques.

    Science.gov (United States)

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M

    2015-07-14

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.

  7. Axonal regeneration in zebrafish spinal cord

    Science.gov (United States)

    Hui, Subhra Prakash

    2018-01-01

    Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

  8. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

    Science.gov (United States)

    Dumoulin, Alexandre; Ter-Avetisyan, Gohar; Schmidt, Hannes; Rathjen, Fritz G

    2018-04-24

    Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  9. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade

    Directory of Open Access Journals (Sweden)

    Alexandre Dumoulin

    2018-04-01

    Full Text Available Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP, the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα. In the absence of any one of these components, neurons in dorsal root ganglia (DRG and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  10. Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

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    Frederickson Martyn

    2010-01-01

    Full Text Available Abstract Background Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. Results We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. Conclusion Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal

  11. Presynaptic protein synthesis required for NT-3-induced long-term synaptic modulation

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    Je H

    2011-01-01

    Full Text Available Abstract Background Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. Results Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR is fused with bacterial gyrase B domain (GyrB-PKR, which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. Conclusions Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner.

  12. Retinal glia promote dorsal root ganglion axon regeneration.

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    Barbara Lorber

    Full Text Available Axon regeneration in the adult central nervous system (CNS is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.

  13. Synthesis of acid-soluble spore proteins by Bacillus subtilis.

    Science.gov (United States)

    Leventhal, J M; Chambliss, G H

    1982-12-01

    The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

  14. A novel ALS-associated variant in UBQLN4 regulates motor axon morphogenesis

    Science.gov (United States)

    Edens, Brittany M; Yan, Jianhua; Miller, Nimrod; Deng, Han-Xiang; Siddique, Teepu; Ma, Yongchao C

    2017-01-01

    The etiological underpinnings of amyotrophic lateral sclerosis (ALS) are complex and incompletely understood, although contributions to pathogenesis by regulators of proteolytic pathways have become increasingly apparent. Here, we present a novel variant in UBQLN4 that is associated with ALS and show that its expression compromises motor axon morphogenesis in mouse motor neurons and in zebrafish. We further demonstrate that the ALS-associated UBQLN4 variant impairs proteasomal function, and identify the Wnt signaling pathway effector beta-catenin as a UBQLN4 substrate. Inhibition of beta-catenin function rescues the UBQLN4 variant-induced motor axon phenotypes. These findings provide a strong link between the regulation of axonal morphogenesis and a new ALS-associated gene variant mediated by protein degradation pathways. DOI: http://dx.doi.org/10.7554/eLife.25453.001 PMID:28463112

  15. One and two-dimensional electrophoresis of fast axonally-transported proteins in rat nerves following acrylamide and 2,5-hexanedione exposure

    International Nuclear Information System (INIS)

    Sickles, D.W.

    1990-01-01

    Transient and repeated deficiencies in protein delivery to the axon are observed following injections of acrylamide (ACR) and 2,5-hexanedione (2,5-HD) (Sickles DW, Neurotoxicology 10: 91;103, 1989; Neurosci Abstr 14:1219, 1988). We have furthered these studies by measuring the effects of single 50 mg/kg ACR and 4 nmole/kg 2,5-HD injections on the quantity of select fast-transported proteins. Proteins were radiolabelled with 3H-leucine injections of the DRG; 1 and 2 dimensional gels were used for separation of the sciatic nerve (9-45mm distal to the ganglion) homogenates. Scintillation counting demonstrated that transport of all proteins studied were affected by both toxicants. Some variation in effect was observed; a direct correlation between molecular weight (r=0.71) and original quantity of radiolabel (r=0.80) with the percent reduction in transport was observed. Some apparent increases in transport of certain proteins were observed on the 2D gels; but this may indicate a change in the isoelectric points of these transported proteins

  16. Studies of axon-glial cell interactions and periaxonal K+ homeostasis--II. The effect of axonal stimulation, cholinergic agents and transport inhibitors on the resistance in series with the axon membrane.

    Science.gov (United States)

    Hassan, S; Lieberman, E M

    1988-06-01

    The small electrical resistance in series with the axon membrane is generally modeled as the intercellular pathway for current flow through the periaxonal glial (Schwann cell) sheath. The series resistance of the medial giant axon of the crayfish, Procambarus clarkii, was found to vary with conditions known to affect the electrical properties of the periaxonal glia. Series resistance was estimated from computer analysed voltage waveforms generated by axial wire-constant current and space clamp techniques. The average series resistance for all axons was 6.2 +/- 0.5 omega cm2 (n = 128). Values ranged between 1 and 30 omega cm2. The series resistance of axons with low resting membrane resistance (less than 1500 omega cm2) increased an average of 30% when stimulated for 45 s to 7 min (50 Hz) whereas the series resistance of high membrane resistance (greater than 1500 omega cm2) axons decreased an average of 10%. Carbachol (10(-7) M) caused the series resistance of low membrane resistance axons to decrease during stimulation but had no effect on high membrane resistance axons. d-Tubocurare (10(-8) M) caused the series resistance of high membrane resistance axons to increase during stimulation but had no effect on low membrane resistance axons. Bumetanide, a Na-K-Cl cotransport inhibitor and low [K+]o, prevented the stimulation-induced increase in series resistance of low membrane resistance axons but had no effect on the high membrane resistance axons. The results suggest that the series resistance of axons varies in response to the activity of the glial K+ uptake mechanisms stimulated by the appearance of K+ in the periaxonal space during action potential generation.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries

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    Oscar A. Carballo-Molina

    2015-02-01

    Full Text Available Damage caused to neural tissue by disease or injury frequently produces a discontinuity in the nervous system. Such damage generates diverse alterations that are commonly permanent, due to the limited regeneration capacity of the adult nervous system, particularly the Central Nervous System (CNS. The cellular reaction to noxious stimulus leads to several events such as the formation of glial and fibrous scars, which inhibit axonal regeneration in both the CNS and the Peripheral Nervous System (PNS. Although in the PNS there is some degree of nerve regeneration, it is common that the growing axons reinnervate incorrect areas, causing mismatches. Providing a permissive substrate for axonal regeneration in combination with delivery systems for the release of molecules, which enhances axonal growth, could increase regeneration and the recovery of functions in the CNS or the PNS. Currently, there are no effective vehicles to supply growth factors or cells to the damaged/diseased nervous system. Hydrogels are polymers that are biodegradable, biocompatible and have the capacity to deliver a large range of molecules in situ. The inclusion of cultured neural cells into hydrogels forming three-dimensional structures allows the formation of synapses and neuronal survival. There is also evidence showing that hydrogels constitute an amenable substrate for axonal growth of endogenous or grafted cells, overcoming the presence of axonal regeneration inhibitory molecules, in both the central and peripheral nervous systems. Recent experiments suggest that hydrogels can carry and deliver several proteins relevant for improving neuronal survival and axonal growth. Although the use of hydrogels is appealing, its effectiveness is still a matter of discussion, and more results are needed to achieve consistent recovery using different parameters. This review also discusses areas of opportunity where hydrogels can be applied, in order to promote axonal regeneration of

  18. Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat

    NARCIS (Netherlands)

    Cao, Y.; Shumsky, J. S.; Sabol, M. A.; Kushner, R. A.; Strittmatter, S.; Hamers, F. P. T.; Lee, D. H. S.; Rabacchi, S. A.; Murray, M.

    2008-01-01

    Objective. The myelin protein Nogo inhibits axon regeneration by binding to its receptor (NgR) on axons. Intrathecal delivery of an NgR antagonist (NEP1-40) promotes growth of injured corticospinal axons and recovery of motor function following a dorsal hemisection. The authors used a similar design

  19. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  20. Bergmann glia and the recognition molecule CHL1 organize GABAergic axons and direct innervation of Purkinje cell dendrites.

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    Fabrice Ango

    2008-04-01

    Full Text Available The geometric and subcellular organization of axon arbors distributes and regulates electrical signaling in neurons and networks, but the underlying mechanisms have remained elusive. In rodent cerebellar cortex, stellate interneurons elaborate characteristic axon arbors that selectively innervate Purkinje cell dendrites and likely regulate dendritic integration. We used GFP BAC transgenic reporter mice to examine the cellular processes and molecular mechanisms underlying the development of stellate cell axons and their innervation pattern. We show that stellate axons are organized and guided towards Purkinje cell dendrites by an intermediate scaffold of Bergmann glial (BG fibers. The L1 family immunoglobulin protein Close Homologue of L1 (CHL1 is localized to apical BG fibers and stellate cells during the development of stellate axon arbors. In the absence of CHL1, stellate axons deviate from BG fibers and show aberrant branching and orientation. Furthermore, synapse formation between aberrant stellate axons and Purkinje dendrites is reduced and cannot be maintained, leading to progressive atrophy of axon terminals. These results establish BG fibers as a guiding scaffold and CHL1 a molecular signal in the organization of stellate axon arbors and in directing their dendritic innervation.

  1. Botulinum toxin's axonal transport from periphery to the spinal cord.

    Science.gov (United States)

    Matak, Ivica; Riederer, Peter; Lacković, Zdravko

    2012-07-01

    Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet - A pilot study.

    Science.gov (United States)

    Jourdan, Marion; Nair, K Sreekumaran; Carter, Rickey E; Schimke, Jill; Ford, G Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

    2015-06-01

    Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. Copyright © 2014. Published by Elsevier Ltd.

  3. Effect of heat stress on the pattern of protein synthesis in wheat endosperm

    International Nuclear Information System (INIS)

    Inwood, W.; Bernardin, J.

    1990-01-01

    The exposure of detached wheat heads (T. aestivum L. cv Cheyenne) to elevated temperatures resulted not only in the induction of a typical set of high and low molecular weight heat shock proteins (hsps), but also in a differential effect on the synthesis of wheat storage proteins in endosperm tissue when monitored by SDS PAGE of 35 S-labeled polypeptides. The synthesis of hsps in the endosperm had a rapid onset, reached a maximum rate within the first 2 hours at 40 degree C, and then steadily decreased during the next four hours. When heads were returned to 25 degree C after 3 hours at 40 degree C, hsp synthesis did not cease abruptly, but gradually declined over the next several hours. High molecular weight glutenin protein synthesis was drastically reduced with the same time course as heat shock protein synthesis was induced at 40 degree C. Conversely, the synthesis of gliadin proteins remained at a high level at 40 degree C. The synthesis rates for glutenin and gliadin proteins remained at low and high levels, respectively, for as long as the elevated temperature was maintained up to 7 hours

  4. Anterograde axonal transport and intercellular transfer of WGA-HRP in trigeminal-innervated sensory receptors of rat incisive papilla.

    Science.gov (United States)

    Chan, K Y; Byers, M R

    1985-04-08

    The ultrastructure and identification of WGA-HRP-labeled sensory receptors in the rat incisive papilla (the most anterior part of hard palate) were studied using semiserial thin sections. Various sensory receptors were organized according to three locations: dome region (ventral), chemosensory corpuscle region (medial to orifice of incisive canal), and lateral labium (apposing the incisive canal). In the dome region, the sensory receptors were localized in three sensory zones that were associated with surface ridges (one medial and two lateral). In each of these zones, intraepithelial receptor axons and Merkel receptors occurred in the epithelium, while simple unencapsulated corpuscles, glomerular-Meissner corpuscles, and incisive (encapsulated) corpuscles occurred in the lamina propria. In the chemosensory corpuscle region, chemosensory corpuscles and intraepithelial receptor axons were located in the epithelium, and incisive corpuscles were present in the lamina propria. In the lateral labium, only intraepithelial receptor axons were prominent. In all these sensory receptors, the preterminal axons and axon terminals were labeled with the tracer protein. In addition, some nonneuronal cells closely associated with the axon terminals were selectively labeled, e.g., terminal Schwann cells, lamellar Schwann cells, Merkel cells, corpuscular basal cells and chemosensory cells. Other adjacent cells were not labeled, e.g., unspecialized epithelial cells, capsular cells, corpuscular sustentacular cells, and fibroblasts. In both labeled axons and cells, WGA-HRP was incorporated into vesicles, tubules, and vacuolar organelles. The specific intercellular transfer of tracer protein may indicate trophic interactions between axon terminals and support cells in sensory receptors. The specific organization of multiple sensory receptors in the rat incisive papilla may provide a useful alternative system for studying somatosensory physiology.

  5. Inhibition of skeletal muscle protein synthesis in septic intra-abdominal abscess

    International Nuclear Information System (INIS)

    Vary, T.C.; Siegel, J.H.; Tall, B.D.; Morris, J.G.; Smith, J.A.

    1988-01-01

    Chronic sepsis is always associated with profound wasting leading to increased release of amino acids from skeletal muscle. Net protein catabolism may be due to decreased rate of synthesis, increased rate of degradation, or both. To determine whether protein synthesis is altered in chronic sepsis, the rate of protein synthesis in vivo was estimated by measuring the incorporation of [ 3 H]-phenylalanine in skeletal muscle protein in a chronic (5-day) septic rat model induced by creation of a stable intra-abdominal abscess using an E. coli + B. fragilis-infected sterile fecal-agar pellet as foreign body nidus. Septic rats failed to gain weight at rates similar to control animals, therefore control animals were weight matched to the septic animals. The skeletal muscle protein content in septic animals was significantly reduced relative to control animals (0.18 +/- 0.01 vs. 0.21 +/- 0.01 mg protein/gm wet wt; p less than 0.02). The rate of incorporation of [ 3 H]-phenylalanine into skeletal muscle protein from control animals was 39 +/- 4 nmole/gm wet wt/hr or a fractional synthetic rate of 5.2 +/- 0.5%/day. In contrast to control animals, the fractional synthetic rate in septic animals (2.6 +/- 0.2%/day) was reduced by 50% compared to control animals (p less than 0.005). The decreased rate of protein synthesis in sepsis was not due to an energy deficit, as high-energy phosphates and ATP/ADP ratio were not altered. This decrease in protein synthesis occurred even though septic animals consumed as much food as control animals

  6. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques

    Science.gov (United States)

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M.

    2015-01-01

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology. PMID:26124111

  7. Dynamics of oligodendrocyte responses to anterograde axonal (Wallerian) and terminal degeneration in normal and TNF-transgenic mice

    DEFF Research Database (Denmark)

    Drøjdahl, Nina; Fenger, Christina; Nielsen, Helle H

    2004-01-01

    degeneration and lesion-induced axonal sprouting in the hippocampal dentate gyrus in TNF-transgenic mice with the response in genetically normal mice. Transectioning of the entorhino-dentate perforant path axonal projection increased hippocampal TNF mRNA expression in both types of mice, but to significantly...... larger levels in the TNF-transgenics. At 5 days after axonal transection, numbers of oligodendrocytes and myelin basic protein (MBP) mRNA expression in the denervated dentate gyrus in TNF-transgenic mice had increased to the same extent as in nontransgenic littermates. At this time, transgenics showed...

  8. Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features.

    Science.gov (United States)

    Curley, Allison A; Arion, Dominique; Volk, David W; Asafu-Adjei, Josephine K; Sampson, Allan R; Fish, Kenneth N; Lewis, David A

    2011-09-01

    Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia. GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.

  9. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    Science.gov (United States)

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  10. Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Dahle, D.B.; Meechan, P.J.; Carpenter, J.G.

    1981-01-01

    Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m -2 . After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [ 14 C]thymidine into acid-insoluble material. A 2.5 or 5.0h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process. (Auth.)

  11. 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.

  12. Motor axon excitability during Wallerian degeneration

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Alvarez, Susana; Krarup, Christian

    2008-01-01

    Axonal loss and degeneration are major factors in determining long-term outcome in patients with peripheral nerve disorders or injury. Following loss of axonal continuity, the isolated nerve stump distal to the lesion undergoes Wallerian degeneration in several phases. In the initial 'latent' phase......, action potential propagation and structural integrity of the distal segment are maintained. The aim of this study was to investigate in vivo the changes in membrane function of motor axons during the 'latent' phase of Wallerian degeneration. Multiple indices of axonal excitability of the tibial nerve...

  13. Npn-1 contributes to axon-axon interactions that differentially control sensory and motor innervation of the limb.

    Directory of Open Access Journals (Sweden)

    Rosa-Eva Huettl

    2011-02-01

    Full Text Available The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1 in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG, we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs.

  14. Protein synthesis by isolated type II pneumocytes in suspension and in primary culture

    International Nuclear Information System (INIS)

    Brandes, M.E.; Finkelstein, J.N.

    1987-01-01

    Protein synthesis in rabbit type II pneumocytes immediately after isolation or during the first 7 days in culture was examined by incorporation of [ 3 H] leucine or [ 35 S]methionine. After a 1h incubation with label, total cellular protein was analyzed by 1 or 2-D PAGE and fluorography. Following isolation, incorporation was limited to a small number of proteins of apparent molecular weight 70kD, 55-60kD, 25kD and 20+22kD which appear to lack cognates in cultured cells. At 3h, these isolation proteins (IPs) account for ∼ 50% of the labeled protein. Pretreatment with actinomycin D abolished synthesis of the IPs suggesting a requirement for active mRNA production. These proteins are actively synthesized during the first 10h following cell isolation. Loss of active synthesis is accompanied by a gradual enhancement in synthesis of other proteins. Actin synthesis, 125 I-EGF binding to cultured type II cells indicate changing receptor number and binding affinity with time in culture

  15. EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

    Czech Academy of Sciences Publication Activity Database

    Eva, R.; Koseki, H.; Kanamarlapudi, V.; Fawcett, James

    2017-01-01

    Roč. 130, č. 21 (2017), s. 3663-3675 ISSN 0021-9533 Institutional support: RVO:68378041 Keywords : axon regeneration * axon transport * neuronal polarisation Subject RIV: FH - Neurology OBOR OECD: Neuroscience s (including psychophysiology Impact factor: 4.431, year: 2016

  16. Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

    International Nuclear Information System (INIS)

    Feldherr, C.M.; Hodges, P.; Paine, P.L.

    1988-01-01

    Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [ 3 H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus

  17. Dynamics of mitochondrial transport in axons

    Directory of Open Access Journals (Sweden)

    Robert Francis Niescier

    2016-05-01

    Full Text Available The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.

  18. The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

    Science.gov (United States)

    Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

    2017-09-06

    In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

  19. Axon Termination, Pruning, and Synaptogenesis in the Giant Fiber System of Drosophila melanogaster Is Promoted by Highwire.

    Science.gov (United States)

    Borgen, Melissa; Rowland, Kimberly; Boerner, Jana; Lloyd, Brandon; Khan, Aruna; Murphey, Rodney

    2017-03-01

    The ubiquitin ligase Highwire has a conserved role in synapse formation. Here, we show that Highwire coordinates several facets of central synapse formation in the Drosophila melanogaster giant fiber system, including axon termination, axon pruning, and synaptic function. Despite the similarities to the fly neuromuscular junction, the role of Highwire and the underlying signaling pathways are distinct in the fly's giant fiber system. During development, branching of the giant fiber presynaptic terminal occurs and, normally, the transient branches are pruned away. However, in highwire mutants these ectopic branches persist, indicating that Highwire promotes axon pruning. highwire mutants also exhibit defects in synaptic function. Highwire promotes axon pruning and synaptic function cell-autonomously by attenuating a mitogen-activated protein kinase pathway including Wallenda, c-Jun N-terminal kinase/Basket, and the transcription factor Jun. We also show a novel role for Highwire in non-cell autonomous promotion of synaptic function from the midline glia. Highwire also regulates axon termination in the giant fibers, as highwire mutant axons exhibit severe overgrowth beyond the pruning defect. This excessive axon growth is increased by manipulating Fos expression in the cells surrounding the giant fiber terminal, suggesting that Fos regulates a trans -synaptic signal that promotes giant fiber axon growth. Copyright © 2017 by the Genetics Society of America.

  20. Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

    Science.gov (United States)

    Jourdan, Marion; Nair, K. Sreekumaran; Carter, Rickey E.; Schimke, Jill; Ford, G. Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

    2015-01-01

    Background and Aims Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. Methods To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 hours. [ring-13C6] phenylalanine and [15N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. Results FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; p=0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Conclusions Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. PMID:24972455

  1. The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals.

    Science.gov (United States)

    Su, Y C; Maurel-Zaffran, C; Treisman, J E; Skolnik, E Y

    2000-07-01

    We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct

  2. Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

    Directory of Open Access Journals (Sweden)

    Hiroaki Mochizuki

    2011-05-01

    Full Text Available Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II, an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM and No distributive disjunction (Nod, remains unaltered. Genetic analyses of kinesin light chain (Klc and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of

  3. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Origins of the protein synthesis cycle

    Science.gov (United States)

    Fox, S. W.

    1981-01-01

    Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

  5. Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

    International Nuclear Information System (INIS)

    Hodge, B.O.; Kream, B.E.

    1988-01-01

    We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of [ 3 H]proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis

  6. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    Science.gov (United States)

    Hursel, Rick; Martens, Eveline A P; Gonnissen, Hanne K J; Hamer, Henrike M; Senden, Joan M G; van Loon, Luc J C; Westerterp-Plantenga, Margriet S

    2015-01-01

    Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;Pprotein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;Psynthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;Pprotein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620). In the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and

  7. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    Directory of Open Access Journals (Sweden)

    Rick Hursel

    Full Text Available Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d or low protein (0.4 g protein/kg/d energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001. Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03, synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01 and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001 were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042

  8. Protective effect of a non specific inflammation on bone marrow protein synthesis in irradiated mice

    International Nuclear Information System (INIS)

    Herodin, F.; Roques, P.; Court, L.

    1988-01-01

    Gamma radiations exert a decrease in mouse bone marrow total protein synthesis. A non-specific inflammatory process induced with polyacrylamide microbeads stimulates spleen and marrow protein synthesis and protects the medullar protein synthesis in irradiated mice [fr

  9. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    Muscle cell culture (L/sub 6/) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 ..mu..M compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of (/sup 3/H) leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using (/sup 3/H) leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 ..mu..M level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.

  10. Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

    International Nuclear Information System (INIS)

    Roeder, R.A.; Thorpe, S.D.; Byers, F.M.; Schelling, G.T.; Gunn, J.M.

    1986-01-01

    Muscle cell culture (L 6 ) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effect of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 μM compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of [ 3 H] leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using [ 3 H] leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes by dexamethasone, but increased in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 μM level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle

  11. Use of self-complementary adeno-associated virus serotype 2 as a tracer for labeling axons: implications for axon regeneration.

    Directory of Open Access Journals (Sweden)

    Yingpeng Liu

    Full Text Available Various types of tracers are available for use in axon regeneration, but they require an extra operational tracer injection, time-consuming immunohistochemical analysis and cause non-specific labeling. Considerable efforts over the past years have explored other methodologies, especially the use of viral vectors, to investigate axon regeneration after injury. Recent studies have demonstrated that self-complementary Adeno-Associated Virus (scAAV induced a high transduction efficiency and faster expression of transgenes. Here, we describe for the first time the use of scAAV2-GFP to label long-projection axons in the corticospinal tract (CST, rubrospinal tract (RST and the central axons of dorsal root ganglion (DRG in the normal and lesioned animal models. We found that scAAV2-GFP could efficiently transduce neurons in the sensorimotor cortex, red nucleus and DRG. Strong GFP expression could be transported anterogradely along the axon to label the numerous axon fibers from CST, RST and central axons of DRG separately. Comparison of the scAAV2 vector with single-stranded (ss AAV2 vector in co-labeled sections showed that the scAAV2 vector induced a faster and stronger transgene expression than the ssAAV2 vector in DRG neurons and their axons. In both spinal cord lesion and dorsal root crush injury models, scAAV-GFP could efficiently label the lesioned and regenerated axons around the lesion cavity and the dorsal root entry zone (DREZ respectively. Further, scAAV2-GFP vector could be combined with traditional tracer to specifically label sensory and motor axons after spinal cord lesion. Thus, we show that using scAAV2-GFP as a tracer is a more effective and efficient way to study axon regeneration following injury.

  12. Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury.

    Science.gov (United States)

    Coulibaly, Aminata P; Isaacson, Lori G

    2016-08-03

    Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Habituation to low or high protein intake does not modulate basal or postprandial muscle protein synthesis rates: a randomized trial.

    Science.gov (United States)

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Kouw, Imre Wk; Wall, Benjamin T; Burd, Nicholas A; de Groot, Lisette Cpgm; van Loon, Luc Jc

    2017-02-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high protein intake (HIGH PRO) on the postprandial muscle protein synthetic response. We assessed the impact of LOW PRO compared with HIGH PRO on basal and postprandial muscle protein synthesis rates after the ingestion of 25 g whey protein. Twenty-four healthy, older men [age: 62 ± 1 y; body mass index (in kg/m 2 ): 25.9 ± 0.4 (mean ± SEM)] participated in a parallel-group randomized trial in which they adapted to either a LOW PRO diet (0.7 g · kg -1 · d -1 ; n = 12) or a HIGH PRO diet (1.5 g · kg -1 · d -1 ; n = 12) for 14 d. On day 15, participants received primed continuous l-[ring- 2 H 5 ]-phenylalanine and l-[1- 13 C]-leucine infusions and ingested 25 g intrinsically l-[1- 13 C]-phenylalanine- and l-[1- 13 C]-leucine-labeled whey protein. Muscle biopsies and blood samples were collected to assess muscle protein synthesis rates as well as dietary protein digestion and absorption kinetics. Plasma leucine concentrations and exogenous phenylalanine appearance rates increased after protein ingestion (P 0.05). Plasma exogenous phenylalanine availability over the 5-h postprandial period was greater after LOW PRO than after HIGH PRO (61% ± 1% compared with 56% ± 2%, respectively; P protein synthesis rates increased from 0.031% ± 0.004% compared with 0.039% ± 0.007%/h in the fasted state to 0.062% ± 0.005% compared with 0.057% ± 0.005%/h in the postprandial state after LOW PRO compared with HIGH PRO, respectively (P protein-derived amino acids in the circulation and does not lower basal muscle protein synthesis rates or increase postprandial muscle protein synthesis rates after ingestion of 25 g protein in older men. This trial was registered at clinicaltrials.gov as NCT

  14. Pαx6 expression in postmitotic neurons mediates the growth of axons in response to SFRP1.

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    Alvaro Sebastián-Serrano

    Full Text Available During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs, dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.

  15. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    International Nuclear Information System (INIS)

    Horst, M.N.

    1990-01-01

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine

  16. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals

    International Nuclear Information System (INIS)

    Woodson, W.R.; Handa, A.K.

    1986-01-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of 3 H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide

  17. p27Kip1 Modulates Axonal Transport by Regulating α-Tubulin Acetyltransferase 1 Stability

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    Giovanni Morelli

    2018-05-01

    Full Text Available Summary: The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27Kip1 controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27Kip1 ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27Kip1 stabilizes the α-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport. : Morelli et al. report that p27Kip1/Dacapo modulates the acetylation of microtubules in axons via stabilization of ATAT1, the main α-tubulin acetyltransferase. Its conditional loss leads to the reduction of bidirectional axonal transport of vesicles and mitochondria in vitro in mice and in vivo in Drosophila. Keywords: p27Kip1, dacapo, acetylation, axonal transport, ATAT1, alpha-tubulin, HDAC6, Drosophila, mouse, cerebral cortex

  18. Fractional cable equation for general geometry: A model of axons with swellings and anomalous diffusion

    Science.gov (United States)

    López-Sánchez, Erick J.; Romero, Juan M.; Yépez-Martínez, Huitzilin

    2017-09-01

    Different experimental studies have reported anomalous diffusion in brain tissues and notably this anomalous diffusion is expressed through fractional derivatives. Axons are important to understand neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease, and Parkinson's disease. Indeed, abnormal accumulation of proteins and organelles in axons is a hallmark of these diseases. The diffusion in the axons can become anomalous as a result of this abnormality. In this case the voltage propagation in axons is affected. Another hallmark of different neurodegenerative diseases is given by discrete swellings along the axon. In order to model the voltage propagation in axons with anomalous diffusion and swellings, in this paper we propose a fractional cable equation for a general geometry. This generalized equation depends on fractional parameters and geometric quantities such as the curvature and torsion of the cable. For a cable with a constant radius we show that the voltage decreases when the fractional effect increases. In cables with swellings we find that when the fractional effect or the swelling radius increases, the voltage decreases. Similar behavior is obtained when the number of swellings and the fractional effect increase. Moreover, we find that when the radius swelling (or the number of swellings) and the fractional effect increase at the same time, the voltage dramatically decreases.

  19. Glucocorticoid effects on hippocampal protein synthesis

    International Nuclear Information System (INIS)

    Schlatter, L.K.

    1988-01-01

    Following subcutaneous injection of rats with 5 mg corticosterone, hippocampal slices in vitro show increased [ 35 S]-methionine labeling of a cytosolic protein with an apparent molecular weight (M r ) of 35,000 and an isoelectric point (IEP) of 6.6. This labeling is temporally consistent with a transcriptional event, and is steroid- and tissue-specific. The pear serum concentration of steroid occurs one hour or less following the injection. Maximal labeling of this protein is reached whenever serum corticosterone values are approximately 100 ng/ml. When endogenous corticosterone levels are elevated to 100 ng/ml through stressors or exogenous ACTH injections the same maximal increase in synthesis of the 35,000 M r protein is observed. Adrenalectomy prevents the observed response from occurring following stressor application or ACTH injections. Comparison of the increases observed after administration of the type 2 receptor agonist RU 28362 and aldosterone, which has a higher affinity for the type 1 receptor, shows a 50-fold greater sensitivity of the response to the type 2 receptor agonist. Synthesis of this protein following serum increases of steroid possibly correlates to the theorized function of the type 2 receptor feedback regulation. The similar protein in the liver has an IEP of 6.8 and a slightly higher M r . A second hippocampal protein with an M r of 46,000 and an IEP of 6.2 is also increased in labeling. Two additional liver proteins, one of Mr 53,000 (IEP of 6.2) and the other with an M r of 45,000 (IEP of 8.7-7.8) are increased in the liver following glucocorticoid administration

  20. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  1. Live Imaging of Calcium Dynamics during Axon Degeneration Reveals Two Functionally Distinct Phases of Calcium Influx

    Science.gov (United States)

    Yamagishi, Yuya; Tessier-Lavigne, Marc

    2015-01-01

    Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program. SIGNIFICANCE STATEMENT Axon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled

  2. Chemical synthesis of membrane proteins by the removable backbone modification method.

    Science.gov (United States)

    Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

    2017-12-01

    Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

  3. Goldberg-Shprintzen megacolon syndrome with associated sensory motor axonal neuropathy.

    Science.gov (United States)

    Dafsari, Hormos Salimi; Byrne, Susan; Lin, Jean-Pierre; Pitt, Matthew; Jongbloed, Jan Dh; Flinter, Frances; Jungbluth, Heinz

    2015-06-01

    Goldberg-Shprintzen megacolon syndrome (GOSHS) (OMIM 609460) is characterized by a combination of learning difficulties, characteristic dysmorphic features and Hirschsprung's disease. Variable clinical features include iris coloboma, congenital heart defects and central nervous system abnormalities, in particular polymicrogyria. GOSHS has been attributed to recessive mutations in KIAA1279, encoding kinesin family member (KIF)-binding protein (KBP) with a crucial role in neuronal microtubule dynamics. Here we report on a 7-year-old girl with GOSHS as a result of a homozygous deletion of exons 5 and 6 of the KIAA1279 gene. She had been referred with the suspicion of an underlying neuromuscular disorder before the genetic diagnosis was established, prompted by the findings of motor developmental delay, hypotonia, ptosis and absent reflexes. Neurophysiological studies revealed unequivocal evidence of a peripheral axonal sensory motor neuropathy. We hypothesize that an axonal sensory motor neuropathy may be part of the phenotypical spectrum of KIAA1279-related GOSHS, probably reflecting the effects of reduced KBP protein expression on peripheral neuronal function. © 2015 Wiley Periodicals, Inc.

  4. Increased slow transport in axons of regenerating newt limbs after a nerve conditioning lesion made prior to amputation

    International Nuclear Information System (INIS)

    Maier, C.E.

    1989-01-01

    The first part of this study shows that axonal density is constant in the limb stump of the next proximal to the area of traumatic nerve degeneration caused by limb amputation. The results of the second part of this work reveal that a nerve conditioning lesion made two weeks prior to amputation is associated with accelerated limb regeneration and that this accelerated limb regeneration is accompanied by an earlier arrival of axons. This is the first demonstration of naturally occurring limb regeneration being enhanced. In this study SCb cytoskeletal proteins were identified and measured using SDS-PAGE and liquid scintillation counting. Proteins were measured at 7, 14, 21, and 28 days after 35 S-methionine injection and the normal rate of SCb transport determined to be 0.19 mm/day. A single axotomy does not enhance the rate of SCb transport but does increase the amount of labeled SCb proteins that are transported. When a conditioning lesion is employed prior to limb amputation and SCb proteins are measured at 7, 14, and 21 days after injection, there is a twofold acceleration in the rate of SCb transport and an increase in the amount of SCb proteins transported in conditioned axons

  5. Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Ana Ferreras

    2002-04-01

    Full Text Available A cell-free system from Plasmodium falciparum able to translate endogenous mRNA was used to determine the effect of artemisinin, chloroquine and primaquine on the protein synthesis mechanism of the parasite. The antimalarial drugs did not inhibit the incorporation of [³H] methionine into parasite proteins even at concentrations higher than the ones found to strongly inhibit the parasite growth. Results clearly indicate that these compounds do not have a direct effect on protein synthesis activity of P. falciparum coded by endogenous mRNA.

  6. Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

    Science.gov (United States)

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

    Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…

  7. Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I

    International Nuclear Information System (INIS)

    Barac-Nieto, M.; Lui, S.M.; Spitzer, A.

    1991-01-01

    Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus? Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy? To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats

  8. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  9. Effect of diet protein quality on growth and protein synthesis in rats

    International Nuclear Information System (INIS)

    Chinchalkar, D.V.; Mehta, S.L.

    1978-01-01

    The effect of diet protein quality on albino rats was studied by feeding normal and opaque-2 maize. The weight gain in rats was 60 percent higher on opaque-2 maize as compared to those fed on normal maize. Rats converted 1.0 g of dietary opaque-2 maize to 0.226 g weight gain as compared to 0.131 g for normal maize. The protein content per liver was higher with opaque-2 maize diet suggesting a higher net protein synthesis in opaque-2 maize fed rat livers. In vitro 14 C-phenylalanine incorporation showed that polysomes from opaque-2 maize fed rat livers were more efficient in protein synthesis than those from normal maize fed rat livers. Addition of poly-U resulted in more enhanced amino acid incorporation with polysomes from normal maize fed rats as compared to other group indicating greater limitation of mRNA in polysomes from normal maize fed rats. The total yield of liver polysomes from opaque-2 maize fed rats was substantially higher. (author)

  10. Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis

    International Nuclear Information System (INIS)

    Krueger, R.J.; Orme-Johnson, N.R.

    1988-01-01

    Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues

  11. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

    Directory of Open Access Journals (Sweden)

    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  12. Chondroitin sulfates do not impede axonal regeneration in goldfish spinal cord.

    Science.gov (United States)

    Takeda, Akihito; Okada, Soichiro; Funakoshi, Kengo

    2017-10-15

    Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Axon guidance pathways served as common targets for human speech/language evolution and related disorders.

    Science.gov (United States)

    Lei, Huimeng; Yan, Zhangming; Sun, Xiaohong; Zhang, Yue; Wang, Jianhong; Ma, Caihong; Xu, Qunyuan; Wang, Rui; Jarvis, Erich D; Sun, Zhirong

    2017-11-01

    Human and several nonhuman species share the rare ability of modifying acoustic and/or syntactic features of sounds produced, i.e. vocal learning, which is the important neurobiological and behavioral substrate of human speech/language. This convergent trait was suggested to be associated with significant genomic convergence and best manifested at the ROBO-SLIT axon guidance pathway. Here we verified the significance of such genomic convergence and assessed its functional relevance to human speech/language using human genetic variation data. In normal human populations, we found the affected amino acid sites were well fixed and accompanied with significantly more associated protein-coding SNPs in the same genes than the rest genes. Diseased individuals with speech/language disorders have significant more low frequency protein coding SNPs but they preferentially occurred outside the affected genes. Such patients' SNPs were enriched in several functional categories including two axon guidance pathways (mediated by netrin and semaphorin) that interact with ROBO-SLITs. Four of the six patients have homozygous missense SNPs on PRAME gene family, one youngest gene family in human lineage, which possibly acts upon retinoic acid receptor signaling, similarly as FOXP2, to modulate axon guidance. Taken together, we suggest the axon guidance pathways (e.g. ROBO-SLIT, PRAME gene family) served as common targets for human speech/language evolution and related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Potential Involvement of Draxin in the Axonal Projection of Cranial Nerves, Especially Cranial Nerve X, in the Chick Hindbrain.

    Science.gov (United States)

    Zhang, Sanbing; Cui, Huixian; Wang, Lei; Kang, Lin; Huang, Guannan; Du, Juan; Li, Sha; Tanaka, Hideaki; Su, Yuhong

    2016-07-01

    The appropriate projection of axons within the nervous system is a crucial component of the establishment of neural circuitry. Draxin is a repulsive axon guidance protein. Draxin has important functions in the guidance of three commissures in the central nervous system and in the migration of neural crest cells and dI3 interneurons in the chick spinal cord. Here, we report that the distribution of the draxin protein and the location of 23C10-positive areas have a strong temporal and spatial correlation. The overexpression of draxin, especially transmembrane draxin, caused 23C10-positive axon bundles to misproject in the dorsal hindbrain. In addition, the overexpression of transmembrane draxin caused abnormal formation of the ganglion crest of the IX and X cranial nerves, misprojection of some anti-human natural killer-1 (HNK-1)-stained structures in the dorsal roof of the hindbrain, and a simultaneous reduction in the efferent nerves of some motoneuron axons inside the hindbrain. Our data reveal that draxin might be involved in the fascicular projection of cranial nerves in the hindbrain. © 2016 The Histochemical Society.

  15. Diversity of Internal Sensory Neuron Axon Projection Patterns Is Controlled by the POU-Domain Protein Pdm3 in Drosophila Larvae.

    Science.gov (United States)

    Qian, Cheng Sam; Kaplow, Margarita; Lee, Jennifer K; Grueber, Wesley B

    2018-02-21

    Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. Here, we characterize the peripheral anatomy, central axon projection, and diversity of td sensory neurons. We provide evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. We identify two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). We identify expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. Our data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis. SIGNIFICANCE STATEMENT How interoceptive sensory circuits develop, including how sensory neurons diversify and target distinct central regions, is still poorly understood, despite the importance of these sensory systems for maintaining physiological homeostasis. Here, we characterize classes of Drosophila internal sensory neurons (td

  16. Protein synthesis, growth and energetics in larval herring (Clupea harengus) at different feeding regimes

    DEFF Research Database (Denmark)

    Houlihan, D F; Pedersen, B H; Steffensen, J F

    1995-01-01

    Rates of growth, protein synthesis and oxygen consumption were measured in herring larvae, Clupea harengus, in order to estimate the contribution that protein synthesis makes to oxygen consumption during rapid growth at 8°C. Protein synthesis rates were determined in larvae 9 to 17 d after hatching....... Larvae were bathed in (3)H phenylalanine for several hours and the free pool and protein-bound phenylalanine specific radioactivities were determined.Fractional rates of protein synthesis increased 5 to 11 fold with feeding after a period of fasting. Efficiencies of retention of synthesized protein were...... approximately 50% during rapid growth. Rapid growth in herring larvae thus appears to be characterized by moderate levels of protein turnover similar to those obtained for larger fish. Increases in growth rate occurred without changes in RNA concentration, i.e., the larvae increased the efficiency of RNA...

  17. Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy

    NARCIS (Netherlands)

    Kole, Maarten H. P.; Letzkus, Johannes J.; Stuart, Greg J.

    2007-01-01

    Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action

  18. Elucidation of axonal transport by radioautography

    International Nuclear Information System (INIS)

    Droz, Bernard.

    1979-01-01

    Radioautography permits to distinguish various pathways within the axons: the axoplasm which includes soluble enzymes and constituents of the cytoskeleton moving with slow axoplasmic flow; the mitochondria which are conveyed as organelles; the smooth endoplasmic reticulum which ensures the fast axonal transport of membrane constituents delivered to axolemma, synaptic vesicles, presynaptic membranes or mitochondria. Furthermore radioautography makes it possible to visualize intercellular exchanges of molecules between axon and glia

  19. Regulation of axonal development by the nuclear protein hindsight (pebbled) in the Drosophila visual system.

    Science.gov (United States)

    Oliva, Carlos; Sierralta, Jimena

    2010-08-15

    The molecules and networks involved in the process of acquisition and maintenance of the form of a mature neuron are not completely known. Using a misexpression screen we identified the gene hindsight as a gene involved in the process of acquisition of the neuronal morphogenesis in the Drosophila adult nervous system. hindsight encodes a transcription factor known for its role in early developmental processes such as embryonic germ band retraction and dorsal closure, as well as in the establishment of cell morphology, planar cell polarity, and epithelial integrity during retinal development. We describe here a novel function for HNT by showing that both loss and gain of function of HNT affects the pathfinding of the photoreceptors axons. By manipulating the timing and level of HNT expression, together with the number of cells manipulated we show here that the function of HNT in axonal guidance is independent of the HNT functions previously reported in retinal cells. Based on genetic interaction experiments we show that part of HNT function in axonal development is exerted through the regulation of genes involved in the dynamics of the actin cytoskeleton. Copyright 2010 Elsevier Inc. All rights reserved.

  20. EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice.

    Directory of Open Access Journals (Sweden)

    Yona Goldshmit

    Full Text Available Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.

  1. 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.

  2. The axonal cytoskeleton : from organization to function

    NARCIS (Netherlands)

    Kevenaar, Josta T; Hoogenraad, Casper C

    The axon is the single long fiber that extends from the neuron and transmits electrical signals away from the cell body. The neuronal cytoskeleton, composed of microtubules (MTs), actin filaments and neurofilaments, is not only required for axon formation and axonal transport but also provides the

  3. Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

    Science.gov (United States)

    Tudor, Jennifer C; Davis, Emily J; Peixoto, Lucia; Wimmer, Mathieu E; van Tilborg, Erik; Park, Alan J; Poplawski, Shane G; Chung, Caroline W; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

    2016-04-26

    Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. Copyright © 2016, American Association for the Advancement of Science.

  4. Solid-phase synthesis of protein-polymers on reversible immobilization supports.

    Science.gov (United States)

    Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

    2018-02-27

    Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

  5. Differential effects of myostatin deficiency on motor and sensory axons.

    Science.gov (United States)

    Jones, Maria R; Villalón, Eric; Northcutt, Adam J; Calcutt, Nigel A; Garcia, Michael L

    2017-12-01

    Deletion of myostatin in mice (MSTN -/- ) alters structural properties of peripheral axons. However, properties like axon diameter and myelin thickness were analyzed in mixed nerves, so it is unclear whether loss of myostatin affects motor, sensory, or both types of axons. Using the MSTN -/- mouse model, we analyzed the effects of increasing the number of muscle fibers on axon diameter, myelin thickness, and internode length in motor and sensory axons. Axon diameter and myelin thickness were increased in motor axons of MSTN -/- mice without affecting internode length or axon number. The number of sensory axons was increased without affecting their structural properties. These results suggest that motor and sensory axons establish structural properties by independent mechanisms. Moreover, in motor axons, instructive cues from the neuromuscular junction may play a role in co-regulating axon diameter and myelin thickness, whereas internode length is established independently. Muscle Nerve 56: E100-E107, 2017. © 2017 Wiley Periodicals, Inc.

  6. A statistical view of protein chemical synthesis using NCL and extended methodologies.

    Science.gov (United States)

    Agouridas, Vangelis; El Mahdi, Ouafâa; Cargoët, Marine; Melnyk, Oleg

    2017-09-15

    Native chemical ligation and extended methodologies are the most popular chemoselective reactions for protein chemical synthesis. Their combination with desulfurization techniques can give access to small or challenging proteins that are exploited in a large variety of research areas. In this report, we have conducted a statistical review of their use for protein chemical synthesis in order to provide a flavor of the recent trends and identify the most popular chemical tools used by protein chemists. To this end, a protein chemical synthesis (PCS) database (http://pcs-db.fr) was created by collecting a set of relevant data from more than 450 publications covering the period 1994-2017. A preliminary account of what this database tells us is presented in this report. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis

    International Nuclear Information System (INIS)

    Ortiz, W.

    1990-01-01

    Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the bleaching temperature of 33 degree C. A primary goal of this work was to establish whether an increase in the synthesis of plastid proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with [ 35 S]sodium sulfate were carried out with cells grown at room temperature or at 33 degree C. The synthesis of a number of plastid polypeptides of nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 33 degree C. In contrast, while synthesis of thylakoid proteins by the plastid protein synthesis machinery decreased modestly, synthesis of the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis of novel plastid-encoded polypeptides was not induced at the bleaching temperature. It is concluded that protein synthesis in plastids declines during the initial phase of the temperature response in Euglena despite an overall increase in cellular protein synthesis and an increase in chlorophyll accumulation per cell

  8. Synthesis and thermotolerance of heat shock proteins in Campylobacter jejuni

    International Nuclear Information System (INIS)

    Kim, C.K.; Kim, H.O.; Lee, K.J.

    1991-01-01

    The heat shock responses of Campylobacter jejuni were studied by examination of their survival rates and synthesis of heat shock proteins. When C. jejuni cells were treated at the sublethal temperatures of 48C° for 30 minutes, most of the cells maintained their viabilities and synthesized the heat shock proteins of 90, 73, and 66 kD in molecular weight. By the method of two-dimensional electrophoresis, the heat shock proteins of C. jejuni were identified to be Hsp90, Hsp73, and Hsp66. During the heat shock at 48C°, the heat shock proteins were induced from about 5 minutes after the heat shock treatment. Their synthesis was continued upto 30 minutes, but remarkably retarded after 50 minutes. When C. jejune cells were heat shocked at 51C° for 30 minutes, the survival rates of the cells were decreased by about 10 3 fold and synthesis of heat shock proteins and normal proteins was also generally retarded. The cells exposed to 55C° for 30 minutes died off by more than 10 5 cells and the new protein synthesis was not observed. But when C. jejuni cells were heat-shocked at the sublethal temperature of 48C° for 15 to 20 minutes and then were exposed at the lethal temperature of 55C° for 30 minutes, their viabilities were higher than those exposed at 55C° for 30 minutes without pre-heat shock at 48C°. Therefore, the heat shock proteins synthesized at the sublethal temperature of 48C° in C. jejuni were thought to be responsible for thermotolerance. However, when C. jejuni cells heat-shocked at various ranges of sublethal and lethal temperatures were placed back to the optimum temperature of 42C°, the multiplication patterns of the cells pretreated at different temperatures were not much different each other

  9. Dendrites of cerebellar granule cells correctly recognize their target axons for synaptogenesis in vitro.

    Science.gov (United States)

    Ito, Shoko; Takeichi, Masatoshi

    2009-08-04

    Neural circuits are generated by precisely ordered synaptic connections among neurons, and this process is thought to rely on the ability of neurons to recognize specific partners. However, it is also known that neurons promiscuously form synapses with nonspecific partners, in particular when cultured in vitro, causing controversies about neural recognition mechanisms. Here we reexamined whether neurons can or cannot select particular partners in vitro. In the cerebellum, granule cell (GC) dendrites form synaptic connections specifically with mossy fibers, but not with climbing fibers. We cocultured GC neurons with pontine or inferior olivary axons, the major sources for mossy and climbing fibers, respectively, as well as with hippocampal axons as a control. The GC neurons formed synapses with pontine axons predominantly at the distal ends of their dendrites, reproducing the characteristic morphology of their synapses observed in vivo, whereas they failed to do so when combined with other axons. In the latter case, synaptic proteins could accumulate between axons and dendrites, but these synapses were randomly distributed throughout the contact sites, and also their synaptic vesicle recycling was anomalous. These observations suggest that GC dendrites can select their authentic partners for synaptogenesis even in vitro, forming the synapses with a GC-specific nature only with them.

  10. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis

    Science.gov (United States)

    Ferrando, A. A.; Lane, H. W.; Stuart, C. A.; Davis-Street, J.; Wolfe, R. R.

    1996-01-01

    We sought to determine the extent to which the loss of lean body mass and nitrogen during inactivity was due to alterations in skeletal muscle protein metabolism. Six male subjects were studied during 7 days of diet stabilization and after 14 days of stimulated microgravity (-6 degrees bed rest). Nitrogen balance became more negative (P protein synthesis (PS; P protein also decreased by 46% (P protein breakdown and inward transport. Whole body protein synthesis determined by [15N]alanine ingestion on six subjects also revealed a 14% decrease (P protein breakdown change significantly. These results indicate that the loss of body protein with inactivity is predominantly due to a decrease in muscle PS and that this decrease is reflected in both whole body and skeletal muscle measures.

  11. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    Science.gov (United States)

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  12. Improved synthesis of (S)-N-Boc-5-oxaproline for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation.

    Science.gov (United States)

    Murar, Claudia E; Harmand, Thibault J; Bode, Jeffrey W

    2017-09-15

    We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate. This route is practical, scalable and economical and provides decagram amounts of material for protein synthesis and conversion to other protected forms of (S)-oxaproline. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Control of protein synthesis in the female pupa of Bombyx mori

    International Nuclear Information System (INIS)

    Yamao, Masami; Koga, Katsumi

    1975-01-01

    For the purpose of understanding the mechanisms of insect metamorphosis, protein synthesis by silkmoth pupae has been studied. Synthetic rate and contents of total RNA and protein changed markedly in the female pupae of Bombyx mori. Attempt was made to find what the limiting step for the synthesis of the bulk of proteins during the adult development of female pupae is. Several female pupae of hydridstrain were homogenized at each of stated periods in buffer. The ribosomal fraction prepared from the homogenates was incubated in the buffer containing 3 H-leucine or 3 H-phenylalanine. The incorporation of leucine depending on endogenous mRNA and that of phenylalanine directed by added poly U were the largest in 9--10 days and 7th day, respectively. From the results, the synthesis of protein during the late adult development of female silkworms is controlled at the level of mRNA. The increase of ribosomes, which were active to bind mRNA, preceded the appearance of available endogenous mRNA, and it may be attributed to neogenesis and ''run-off'' of previous ribosomes. It is conceivable that such neogenesis or run-off serves as less direct control for the protein synthesis during the metamorphosis of Bombix mori. (Kobatake, H.)

  14. Neurogenetics of slow axonal transport: from cells to animals.

    Science.gov (United States)

    Sadananda, Aparna; Ray, Krishanu

    2012-09-01

    Slow axonal transport is a multivariate phenomenon implicated in several neurodegenerative disorders. Recent reports have unraveled the molecular basis of the transport of certain slow component proteins, such as the neurofilament subunits, tubulin, and certain soluble enzymes such as Ca(2+)/calmodulin-dependent protein kinase IIa (CaM kinase IIa), etc., in tissue cultured neurons. In addition, genetic analyses also implicate microtubule-dependent motors and other housekeeping proteins in this process. However, the biological relevance of this phenomenon is not so well understood. Here, the authors have discussed the possibility of adopting neurogenetic analyses in multiple model organisms to correlate molecular level measurements of the slow transport phenomenon to animal behavior, thus facilitating the investigation of its biological efficacy.

  15. Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

    Science.gov (United States)

    Walsh, Derek; Mathews, Michael B.; Mohr, Ian

    2013-01-01

    Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

  16. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis

    Science.gov (United States)

    Zambonin, Jessica L.; Zhao, Chao; Ohno, Nobuhiko; Campbell, Graham R.; Engeham, Sarah; Ziabreva, Iryna; Schwarz, Nadine; Lee, Sok Ee; Frischer, Josa M.; Turnbull, Doug M.; Trapp, Bruce D.; Lassmann, Hans; Franklin, Robin J. M.

    2011-01-01

    Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in

  17. Creatine pretreatment protects cortical axons from energy depletion in vitro

    Science.gov (United States)

    Shen, Hua; Goldberg, Mark P.

    2012-01-01

    Creatine is a natural nitrogenous guanidino compound involved in bioenergy metabolism. Although creatine has been shown to protect neurons of the central nervous system (CNS) from experimental hypoxia/ischemia, it remains unclear if creatine may also protect CNS axons, and if the potential axonal protection depends on glial cells. To evaluate the direct impact of creatine on CNS axons, cortical axons were cultured in a separate compartment from their somas and proximal neurites using a modified two-compartment culture device. Axons in the axon compartment were subjected to acute energy depletion, an in vitro model of white matter ischemia, by exposure to 6 mM sodium azide for 30 min in the absence of glucose and pyruvate. Energy depletion reduced axonal ATP by 65%, depolarized axonal resting potential, and damaged 75% of axons. Application of creatine (10 mM) to both compartments of the culture at 24 h prior to energy depletion significantly reduced axonal damage by 50%. In line with the role of creatine in the bioenergy metabolism, this application also alleviated the axonal ATP loss and depolarization. Inhibition of axonal depolarization by blocking sodium influx with tetrodotoxin also effectively reduced the axonal damage caused by energy depletion. Further study revealed that the creatine effect was independent of glial cells, as axonal protection was sustained even when creatine was applied only to the axon compartment (free from somas and glial cells) for as little as 2 h. In contrast, application of creatine after energy depletion did not protect axons. The data provide the first evidence that creatine pretreatment may directly protect CNS axons from energy deficiency. PMID:22521466

  18. Selective memory generalization by spatial patterning of protein synthesis.

    Science.gov (United States)

    O'Donnell, Cian; Sejnowski, Terrence J

    2014-04-16

    Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings, we proposed a two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

    International Nuclear Information System (INIS)

    Gailit, James

    1990-01-01

    Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

  20. Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, J E; Sandberg, M [Department of Neurology, University Hospital, S-221 85 Lund, Sweden

    1975-01-01

    The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of /sup 14/C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed.

  1. Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

    International Nuclear Information System (INIS)

    Olsson, J.-E.; Sandberg, M.

    1975-01-01

    The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of 14 C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed. (author)

  2. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    Science.gov (United States)

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. ENA/VASP downregulation triggers cell death by impairing axonal maintenance in hippocampal neurons.

    Science.gov (United States)

    Franco, D Lorena; Rezával, Carolina; Cáceres, Alfredo; Schinder, Alejandro F; Ceriani, M Fernanda

    2010-06-01

    Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration. 2010 Elsevier Inc. All rights reserved.

  4. Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

    Science.gov (United States)

    Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

    2015-09-01

    Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

    Science.gov (United States)

    Kleihues, P.; Magee, P. N.

    1973-01-01

    1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA. PMID:4774397

  6. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  7. mTORC1-independent reduction of retinal protein synthesis in type 1 diabetes.

    Science.gov (United States)

    Fort, Patrice E; Losiewicz, Mandy K; Pennathur, Subramaniam; Jefferson, Leonard S; Kimball, Scot R; Abcouwer, Steven F; Gardner, Thomas W

    2014-09-01

    Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2(Akita) diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  8. Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis I. The effect of ribonuclease on protein synthesis

    NARCIS (Netherlands)

    Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

    1961-01-01

    Ribonuclease was found to inhibit the protein synthesis in the naked yeast protoplast for nearly 100%. Even small concentrations (5 μg/ml) were found inhibitory. The cause of this inhibition can be attributed at least in part to a 90% inhibition of the respiration. Amino acid uptake was found to

  9. Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

    Science.gov (United States)

    In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

  10. Schwann Cell Glycogen Selectively Supports Myelinated Axon Function

    Science.gov (United States)

    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

  11. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    International Nuclear Information System (INIS)

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 μM) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, 14 C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively. (author)

  12. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    Energy Technology Data Exchange (ETDEWEB)

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 ..mu..M) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, /sup 14/C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively.

  13. The limits of adaptation of functional protein synthesis to sever undernutrition

    International Nuclear Information System (INIS)

    Jahoor, F.; Bhattiprolu, S.; Reeds, P.; Forrester, T.; Boyne, M.

    1994-01-01

    Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child's prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab

  14. The limits of adaptation of functional protein synthesis to sever undernutrition

    Energy Technology Data Exchange (ETDEWEB)

    Jahoor, F; Bhattiprolu, S; Reeds, P [Baylor Coll. of Medicine, Houston, TX (United States). Children` s Nutrition Research Centre; Forrester, T; Boyne, M [West Indies Univ., Mona (Jamaica). Tropical Metabolism Research Unit

    1994-12-31

    Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child`s prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab.

  15. Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

    International Nuclear Information System (INIS)

    Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

    1984-01-01

    Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)

  16. Muscle and liver protein synthesis in growing rats fed diets containing raw legumes as the main source of protein

    International Nuclear Information System (INIS)

    Goena, M.; Santidrian, S.; Cuevillas, F.; Larralde, J.

    1986-01-01

    Although legumes are widely used as protein sources, their effects on protein metabolism remain quite unexplored. The authors have measured the rates of gastrocnemius muscle and liver protein synthesis in growing rats fed ad libitum over periods of 12 days on diets containing raw field bean (Vicia faba L.), raw kidney bean (Phaseolus vulgaris L.), and raw bitter vetch (Vicia ervilia L.) as the major sources of protein. Diets were isocaloric and contained about 12% protein. Protein synthesis was evaluated by the constant-intravenous-infusion method, using L-/ 14 C/-tyrosine, as well as by the determination of the RNA-activity (g of newly synthesized protein/day/g RNA). Results showed that, as compared to well-fed control animals, those fed the raw legume diets exhibited a marked reduction in the rate of growth with no changes in the amount of food intake (per 100 g b.wt.). These changes were accompanied by a significant reduction in the rate of muscle protein synthesis in all legume-treated rats, being this reduction greater in the animals fed the Ph. vulgaris and V. ervilia diets. Liver protein synthesis was slightly higher in the rats fed the V. faba and V. ervilia diets, and smaller in the Ph. vulgaris-fed rats. It is suggested that both sulfur amino acid deficiency and the presence of different anti-nutritive factors in raw legumes may account for these effects

  17. Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

    International Nuclear Information System (INIS)

    Sardinia, L.M.

    1985-01-01

    The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with 35 S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with 32 P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships

  18. Regulation of Adult CNS Axonal Regeneration by the Post-transcriptional Regulator Cpeb1

    Directory of Open Access Journals (Sweden)

    Wilson Pak-Kin Lou

    2018-01-01

    Full Text Available Adult mammalian central nervous system (CNS neurons are unable to regenerate following axonal injury, leading to permanent functional impairments. Yet, the reasons underlying this regeneration failure are not fully understood. Here, we studied the transcriptome and translatome shortly after spinal cord injury. Profiling of the total and ribosome-bound RNA in injured and naïve spinal cords identified a substantial post-transcriptional regulation of gene expression. In particular, transcripts associated with nervous system development were down-regulated in the total RNA fraction while remaining stably loaded onto ribosomes. Interestingly, motif association analysis of post-transcriptionally regulated transcripts identified the cytoplasmic polyadenylation element (CPE as enriched in a subset of these transcripts that was more resistant to injury-induced reduction at the transcriptome level. Modulation of these transcripts by overexpression of the CPE binding protein, Cpeb1, in mouse and Drosophila CNS neurons promoted axonal regeneration following injury. Our study uncovered a global evolutionarily conserved post-transcriptional mechanism enhancing regeneration of injured CNS axons.

  19. Predictors of muscle protein synthesis after severe pediatric burns

    Science.gov (United States)

    Objectives: Following a major burn, muscle protein synthesis rate increases but in most patients, this response is not sufficient to compensate the also elevated protein breakdown. Given the long-term nature of the pathophysiologic response to burn injury, we hypothesized that skeletal muscle prot...

  20. Pathogenesis of axonal dystrophy and demyelination in alphaA-crystallin-expressing transgenic mice.

    NARCIS (Netherlands)

    Rijk, A. van; Sweers, M.A.; Merkx, G.F.M.; Lammens, M.M.Y.; Bloemendal, H.

    2003-01-01

    We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous

  1. Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis.

    Science.gov (United States)

    Inda, Mari Carmen; Delgado-García, José María; Carrión, Angel Manuel

    2005-02-23

    Memory, as measured by changes in an animal's behavior some time after learning, is a reflection of many processes. Here, using a trace paradigm, in mice we show that de novo protein synthesis is required for acquisition, consolidation, reconsolidation, and extinction of classically conditioned eyelid responses. Two critical periods of protein synthesis have been found: the first, during training, the blocking of which impaired acquisition; and the second, lasting the first 4 h after training, the blocking of which impaired consolidation. The process of reconsolidation was sensitive to protein synthesis inhibition if anisomycin was injected before or just after the reactivation session. Furthermore, extinction was also dependent on protein synthesis, following the same temporal course as that followed during acquisition and consolidation. This last fact reinforces the idea that extinction is an active learning process rather than a passive event of forgetting. Together, these findings demonstrate that all of the different stages of memory formation involved in the classical conditioning of eyelid responses are dependent on protein synthesis.

  2. Dynamic changes of the early protein synthesis in murine immune cells after low dose radiation

    International Nuclear Information System (INIS)

    Chen Shali; Liu Shuzheng

    1997-01-01

    It was shown that there was a marked increase in protein synthesis of thymocytes that were metabolically labelled with 3 H-Leu for 4,6,8 and 12 hours in low dose irradiated mice showing 33.26%, 51.48%, 51.54% and 34.98% increase respectively at different time intervals of incubation when the thymic and splenic cells were sampled 4 hours after whole body irradiation (WBI) with 75 mGy X-rays. The results suggest that there is an increase in protein synthesis with its peak at 6∼8 hours after radiation. Changes in protein synthesis of immune cells in mice 4 hours after radiation and incubated for 4∼12 h were observed with SDS-PAGE followed by densitometrical scanning. It is revealed that 28 kD protein synthesis was increased gradually within 12 hours of incubation and 43 kD protein synthesis was increased in the thymocytes rapidly reaching a maximum 2 hours after incubation. It was also exhibited that the synthesis of 43 kD protein and 32 kD protein was increased in the splenocytes 2 hours after incubation. These findings may have implications in the mechanism of immunoenhancement and adaptive response induced by low dose radiation

  3. Death Receptor 6 Promotes Wallerian Degeneration in Peripheral Axons.

    Science.gov (United States)

    Gamage, Kanchana K; Cheng, Irene; Park, Rachel E; Karim, Mardeen S; Edamura, Kazusa; Hughes, Christopher; Spano, Anthony J; Erisir, Alev; Deppmann, Christopher D

    2017-03-20

    Axon degeneration during development is required to sculpt a functional nervous system and is also a hallmark of pathological insult, such as injury [1, 2]. Despite similar morphological characteristics, very little overlap in molecular mechanisms has been reported between pathological and developmental degeneration [3-5]. In the peripheral nervous system (PNS), developmental axon pruning relies on receptor-mediated extrinsic degeneration mechanisms to determine which axons are maintained or degenerated [5-7]. Receptors have not been implicated in Wallerian axon degeneration; instead, axon autonomous, intrinsic mechanisms are thought to be the primary driver for this type of axon disintegration [8-10]. Here we survey the role of neuronally expressed, paralogous tumor necrosis factor receptor super family (TNFRSF) members in Wallerian degeneration. We find that an orphan receptor, death receptor 6 (DR6), is required to drive axon degeneration after axotomy in sympathetic and sensory neurons cultured in microfluidic devices. We sought to validate these in vitro findings in vivo using a transected sciatic nerve model. Consistent with the in vitro findings, DR6 -/- animals displayed preserved axons up to 4 weeks after injury. In contrast to phenotypes observed in Wld s and Sarm1 -/- mice, preserved axons in DR6 -/- animals display profound myelin remodeling. This indicates that deterioration of axons and myelin after axotomy are mechanistically distinct processes. Finally, we find that JNK signaling after injury requires DR6, suggesting a link between this novel extrinsic pathway and the axon autonomous, intrinsic pathways that have become established for Wallerian degeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Towards single-molecule observation of protein synthesis

    International Nuclear Information System (INIS)

    Dulin, David; Le Gall, Antoine; Bouyer, Philippe; Perronet, Karen; Westbrook, Nathalie; Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2009-01-01

    The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, a study at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the development of an advanced surface chemistry to attach an active ribosome to the microscope coverslip and follow the amino-acid incorporation by fluorescence microscopy. The ribosome is labeled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. It should then be possible to observe the protein synthesis in real time as the labeled amino acids are incorporated into the polypeptide chain. (Author)

  5. Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

    Science.gov (United States)

    Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

    2015-09-11

    Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

  6. Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

    Science.gov (United States)

    Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

    2011-01-21

    Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.

  7. Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

    Science.gov (United States)

    Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

    2017-08-01

    Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

  8. Skeletal muscle morphology, protein synthesis and gene expression in Ehlers Danlos Syndrome

    DEFF Research Database (Denmark)

    Nygaard, Rie H; Jensen, Jacob K; Voermans, Nicol C

    2017-01-01

    skeletal muscle biopsies in patients with classic EDS (cEDS, n=5 (Denmark)+ 8 (The Netherlands)) and vascular EDS (vEDS, n=3) and analyzed muscle fiber morphology and content (Western blotting and muscle fiber type/area distributions) and muscle mRNA expression and protein synthesis rate (RT-PCR and stable...... isotope technique). RESULTS: The cEDS patients did not differ from healthy controls (n = 7-11) with regard to muscle fiber type/area, myosin/α-actin ratio, muscle protein synthesis rate or mRNA expression. In contrast, the vEDS patients demonstrated higher expression of matrix proteins compared to c......EDS patients (fibronectin and MMP-2). DISCUSSION: The cEDS patients had surprisingly normal muscle morphology and protein synthesis, whereas vEDS patients demonstrated higher mRNA expression for extracellular matrix remodeling in skeletal musculature compared to cEDS patients....

  9. Electrophysiology of Axonal Constrictions

    Science.gov (United States)

    Johnson, Christopher; Jung, Peter; Brown, Anthony

    2013-03-01

    Axons of myelinated neurons are constricted at the nodes of Ranvier, where they are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions are generated by local regulation of the kinetics of neurofilaments the most important cytoskeletal elements of the axon. In this paper we discuss how this shape affects the electrophysiological function of the neuron. Specifically, although the nodes are short (about 1 μm) in comparison to the distance between nodes (hundreds of μm) they have a substantial influence on the conduction velocity of neurons. We show through computational modeling that nodal constrictions (all other features such as numbers of ion channels left constant) reduce the required fiber diameter for a given target conduction velocity by up to 50% in comparison to an unconstricted axon. We further show that the predicted optimal fiber morphologies closely match reported fiber morphologies. Supported by The National Science Foundation (IOS 1146789)

  10. Tau protein and adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Almudena eFuster-Matanzo

    2012-07-01

    Full Text Available Tau protein is a microtubule associated protein found in the axonal compartment that stabilizes neuronal microtubules under normal physiological conditions. Tau metabolism has attracted much attention because of its role in neurodegenerative disorders called tauopathies, mainly Alzheimer disease. Here, we review recent findings suggesting that axonal outgrowth in subgranular zone during adult hippocampal neurogenesis requires a dynamic microtubule network and tau protein facilitates to maintain that dynamic cytoskeleton. Those functions are carried out in part by tau isoform with only three microtubule-binding domains (without exon 10 and by presence of hypherphosphorylated tau forms. Thus, tau is a good marker and a valuable tool to study new axons in adult neurogenesis.

  11. Internodal function in normal and regenerated mammalian axons

    DEFF Research Database (Denmark)

    Moldovan, M; Krarup, C

    2007-01-01

    AIM: Following Wallerian degeneration, peripheral myelinated axons have the ability to regenerate and, given a proper pathway, establish functional connections with targets. In spite of this capacity, the clinical outcome of nerve regeneration remains unsatisfactory. Early studies have found...... that regenerated internodes remain persistently short though this abnormality did not seem to influence recovery in conduction. It remains unclear to which extent abnormalities in axonal function itself may contribute to the poor outcome of nerve regeneration. METHODS: We review experimental evidence indicating...... that internodes play an active role in axonal function. RESULTS: By investigating internodal contribution to axonal excitability we have found evidence that axonal function may be permanently compromised in regenerated nerves. Furthermore, we illustrate that internodal function is also abnormal in regenerated...

  12. Ribosomal history reveals origins of modern protein synthesis.

    Directory of Open Access Journals (Sweden)

    Ajith Harish

    Full Text Available The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17 and the oldest substructure (the ribosomal ratchet in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  13. SAD kinases sculpt axonal arbors of sensory neurons through long and short-term responses to neurotrophin signals

    Science.gov (United States)

    Lilley, Brendan N.; Pan, Y. Albert; Sanes, Joshua R.

    2013-01-01

    SUMMARY Extrinsic cues activate intrinsic signaling mechanisms to pattern neuronal shape and connectivity. We showed previously that three cytoplasmic Ser/Thr kinases, LKB1, SAD-A and SAD-B, control early axon-dendrite polarization in forebrain neurons. Here we assess their role in other neuronal types. We found that all three kinases are dispensable for axon formation outside of the cortex, but that SAD kinases are required for formation of central axonal arbors by subsets of sensory neurons. The requirement for SAD kinases is most prominent in NT-3 dependent neurons. SAD kinases transduce NT-3 signals in two ways through distinct pathways. First, sustained NT-3/TrkC signaling increases SAD protein levels. Second, short duration NT-3/TrkC signals transiently activate SADs by inducing dephosphorylation of C-terminal domains, thereby allowing activating phosphorylation of the kinase domain. We propose that SAD kinases integrate long- and short duration signals from extrinsic cues to sculpt axon arbors within the CNS. PMID:23790753

  14. Optofluidic control of axonal guidance

    Science.gov (United States)

    Gu, Ling; Ordonez, Simon; Black, Bryan; Mohanty, Samarendra K.

    2013-03-01

    Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc) methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite particles were placed separately in one chamber and single particle was transported through microfluidic channel to another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.

  15. Can injured adult CNS axons regenerate by recapitulating development?

    Science.gov (United States)

    Hilton, Brett J; Bradke, Frank

    2017-10-01

    In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS. © 2017. Published by The Company of Biologists Ltd.

  16. Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation

    International Nuclear Information System (INIS)

    Smith, C.B.; Deibler, G.E.; Eng, N.; Schmidt, K.; Sokoloff, L.

    1988-01-01

    A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1- 14 C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account

  17. Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

    Science.gov (United States)

    Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

    2007-11-01

    In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

  18. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells

    International Nuclear Information System (INIS)

    Santoro, M.G.; Garaci, E.; Amici, C.

    1989-01-01

    Prostaglandins (PGs)A 1 and J 2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA 1 and PGJ 2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ 2 treatment. p74 was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production

  19. Dependence of regenerated sensory axons on continuous neurotrophin-3 delivery.

    Science.gov (United States)

    Hou, Shaoping; Nicholson, LaShae; van Niekerk, Erna; Motsch, Melanie; Blesch, Armin

    2012-09-19

    Previous studies have shown that injured dorsal column sensory axons extend across a spinal cord lesion site if axons are guided by a gradient of neurotrophin-3 (NT-3) rostral to the lesion. Here we examined whether continuous NT-3 delivery is necessary to sustain regenerated axons in the injured spinal cord. Using tetracycline-regulated (tet-off) lentiviral gene delivery, NT-3 expression was tightly controlled by doxycycline administration. To examine axon growth responses to regulated NT-3 expression, adult rats underwent a C3 dorsal funiculus lesion. The lesion site was filled with bone marrow stromal cells, tet-off-NT-3 virus was injected rostral to the lesion site, and the intrinsic growth capacity of sensory neurons was activated by a conditioning lesion. When NT-3 gene expression was turned on, cholera toxin β-subunit-labeled sensory axons regenerated into and beyond the lesion/graft site. Surprisingly, the number of regenerated axons significantly declined when NT-3 expression was turned off, whereas continued NT-3 expression sustained regenerated axons. Quantification of axon numbers beyond the lesion demonstrated a significant decline of axon growth in animals with transient NT-3 expression, only some axons that had regenerated over longer distance were sustained. Regenerated axons were located in white matter and did not form axodendritic synapses but expressed presynaptic markers when closely associated with NG2-labeled cells. A decline in axon density was also observed within cellular grafts after NT-3 expression was turned off possibly via reduction in L1 and laminin expression in Schwann cells. Thus, multiple mechanisms underlie the inability of transient NT-3 expression to fully sustain regenerated sensory axons.

  20. Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

    Science.gov (United States)

    Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

    1978-09-01

    Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

  1. A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

    Science.gov (United States)

    Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

    2017-01-01

    The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the

  2. Glycoproteins of axonal transport: affinity chromatography on fucose-specific lectins

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, S.; Ohlson, C.; Karlsson, J.O.

    1982-03-01

    Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.

  3. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  4. Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

    International Nuclear Information System (INIS)

    Heidenreich, K.A.; Toledo, S.P.

    1989-01-01

    In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of [3H]leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of [3H]aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons

  5. Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

    Science.gov (United States)

    Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

    2017-07-01

    The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  6. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

    Science.gov (United States)

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session.

  7. Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

    Science.gov (United States)

    Hudson, Matthew B; Smuder, Ashley J; Nelson, W Bradley; Wiggs, Michael P; Shimkus, Kevin L; Fluckey, James D; Szeto, Hazel H; Powers, Scott K

    2015-01-01

    Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

  8. Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Matthew B Hudson

    Full Text Available Mechanical ventilation (MV is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1 determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2 establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

  9. Biomarker evidence of axonal injury in neuroasymptomatic HIV-1 patients.

    Directory of Open Access Journals (Sweden)

    Jan Jessen Krut

    Full Text Available Prevalence of neurocognitive impairment in HIV-1 infected patients is reported to be high. Whether this is a result of active HIV-related neurodegeneration is unclear. We examined axonal injury in HIV-1 patients by measuring the light subunit of neurofilament protein (NFL in CSF with a novel, sensitive method.With a cross-sectional design, CSF concentrations of neurofilament protein light (NFL (marker of neuronal injury, neopterin (intrathecal immunoactivation and CSF/Plasma albumin ratio (blood-brain barrier integrity were analyzed on CSF from 252 HIV-infected patients, subdivided into untreated neuroasymptomatics (n = 200, HIV-associated dementia (HAD (n = 14 and on combinations antiretroviral treatment (cART (n = 85, and healthy controls (n = 204. 46 HIV-infected patients were included in both treated and untreated groups, but sampled at different timepoints. Furthermore, 78 neuroasymptomatic patients were analyzed before and after treatment initiation.While HAD patients had the highest NFL concentrations, elevated CSF NFL was also found in 33% of untreated neuroasymptomatic patients, mainly in those with blood CD4+ cell counts below 250 cells/μL. CSF NFL concentrations in the untreated neuroasymptomatics and treated groups were equivalent to controls 18.5 and 3.9 years older, respectively. Neopterin correlated with NFL levels in untreated groups while the albumin ratio correlated with NFL in both untreated and treated groups.Increased CSF NFL indicates ongoing axonal injury in many neuroasymptomatic patients. Treatment decreases NFL, but treated patients retain higher levels than controls, indicating either continued virus-related injury or an aging-like effect of HIV infection. NFL correlates with neopterin and albumin ratio, suggesting an association between axonal injury, neuroinflammation and blood-brain barrier permeability. NFL appears to be a sensitive biomarker of subclinical and clinical brain injury in HIV and warrants further

  10. ROLE OF NEUROTRANSMITTERS AND PROTEIN SYNTHESIS IN SHORT- AND LONG-TERM MEMORY

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, E.L.; Rosenzweig, M.R.; Flood, J.F.

    1978-10-01

    Anisomycin is an effective inhibitor of cerebral protein synthesis in mice and is also an effective amnestic agent for both passive and active behavioral tasks. From use of anisomycin in combination with a variety of stimulant and depressant drugs, we conclude that the level of arousal following acquisition plays an important role in determining the duration and the rate of the biosynthetic phase of memory formation. While we have interpreted the experiments with anisomycin as evidence for an essential role of protein in memory storage, others have suggested that side effects of inhibitors of protein synthesis on catecholamine metabolism are the main cause of amnesia. Several experiments were therefore done to compare the effects of anisemycin and catecholamine inhibitors on memory. We conclude that anisomycin's principal amnestic mechanism does not involve inhibition of the catecholamine system. The results strengthen our conclusion that protein synthesis is an essential component for longterm memory trace formation. Also, it is suggested that proteins synthesized in the neuronal cell body are used, in conjunction with other molecules, to produce permanent and semi-permanent anatomical changes.

  11. 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

  12. 4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats

    Energy Technology Data Exchange (ETDEWEB)

    Lindquist, T D; Ingoglia, N A; Gould, R M [Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, NJ, USA

    1982-12-28

    Experiments were designed to determine if following injection of (/sup 3/H)uridine into the lumbar spinal cord of the rat, (/sup 3/H)RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant predominant RNA species present in these axons.

  13. Protein synthesis levels are increased in a subset of individuals with Fragile X syndrome

    DEFF Research Database (Denmark)

    Jacquemont, Sébastien; Pacini, Laura; Jønch, Aia E

    2018-01-01

    architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical...... severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS...... and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those...

  14. Guidance of retinal axons in mammals.

    Science.gov (United States)

    Herrera, Eloísa; Erskine, Lynda; Morenilla-Palao, Cruz

    2017-11-26

    In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face 'choices' in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Cell-free protein synthesis: applications in proteomics and biotechnology.

    Science.gov (United States)

    He, Mingyue

    2008-01-01

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

  16. Clofibrate-induced increases in peroxisomal proteins: effect on synthesis, degradation, and mRNA activity

    International Nuclear Information System (INIS)

    Mortensen, R.M.

    1983-01-01

    The effect of clofibrate on the polypeptide composition of peroxisomes was determined. A simple method was developed for the isolation of peroxisomes with a purity of 90-95% using sedimentation in a metrizamide gradient. The specific activities of HD did not change with clofibrate treatment so that the increases in enzyme activities are solely due to increases in protein amounts. The hepatic concentration of HD increased 63 times. The HD synthesis rate, as measured by the incorporation of [ 3 H]leucine, increased 74 times, so that the increase in the synthesis was sufficient to account for the increase in protein. Clofibrate caused no discernible change in the degradation rate of HD labeled with [ 14 C]bicarbonate. The half-life of HD was approximately 2 days. The translatable mRBA coding for HD increased 55 times. This value is not significantly different from the increase in HD protein or in HD synthesis. This observation was also true for several other peroxisomal proteins. Therefore, clofibrate causes an increase in the mRNA activity, which increases the synthesis of HD leading to an accumulation of protein and enzyme activity. The kinetics of the clofibrate-induced changes in HD synthesis rate, protein level, and enzymatic activity was analyzed using a simple model which included the half-lives of the drug, mRNA, and protein. The best fit of the model to the data gave an mRNA half-life of 10 hours and a protein half-life of 1.8 days, with no significant change by clofibrate

  17. Safe taste memory consolidation is disrupted by a protein synthesis inhibitor in the nucleus accumbens shell.

    Science.gov (United States)

    Pedroza-Llinás, R; Ramírez-Lugo, L; Guzmán-Ramos, K; Zavala-Vega, S; Bermúdez-Rattoni, F

    2009-07-01

    Consolidation is the process by which a new memory is stabilized over time, and is dependent on de novo protein synthesis. A useful model for studying memory formation is gustatory memory, a type of memory in which a novel taste may become either safe by not being followed by negative consequences (attenuation of neophobia, AN), or aversive by being followed by post-digestive malaise (conditioned taste aversion, CTA). Here we evaluated the effects of the administration of a protein synthesis inhibitor in the nucleus accumbens (NAc) shell for either safe or aversive taste memory trace consolidation. To test the effects on CTA and AN of protein synthesis inhibition, anisomycin (100microg/microl) was bilaterally infused into the NAc shell of Wistar rats' brains. We found that post-trial protein synthesis blockade impaired the long-term safe taste memory. However, protein synthesis inhibition failed to disrupt the long-term memory of CTA. In addition, we infused anisomycin in the NAc shell after the pre-exposure to saccharin in a latent inhibition of aversive taste. We found that the protein synthesis inhibition impaired the consolidation of safe taste memory, allowing the aversive taste memory to form and consolidate. Our results suggest that protein synthesis is required in the NAc shell for consolidation of safe but not aversive taste memories, supporting the notion that consolidation of taste memory is processed in several brain regions in parallel, and implying that inhibitory interactions between both taste memory traces do occur.

  18. Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons.

    Science.gov (United States)

    Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J; Levitan, Edwin S; Wu, Chunlai

    2014-06-18

    During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B' [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). Copyright © 2014 the authors 0270-6474/14/348474-14$15.00/0.

  19. Drosophila Syd-1, Liprin-α, and Protein Phosphatase 2A B′ Subunit Wrd Function in a Linear Pathway to Prevent Ectopic Accumulation of Synaptic Materials in Distal Axons

    Science.gov (United States)

    Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A.; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J.; Levitan, Edwin S.

    2014-01-01

    During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B′ [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). PMID:24948803

  20. Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

    Science.gov (United States)

    Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

    2017-05-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P protein supply seem to

  1. Axonal Conduction Delays, Brain State, and Corticogeniculate Communication.

    Science.gov (United States)

    Stoelzel, Carl R; Bereshpolova, Yulia; Alonso, Jose-Manuel; Swadlow, Harvey A

    2017-06-28

    Thalamocortical conduction times are short, but layer 6 corticothalamic axons display an enormous range of conduction times, some exceeding 40-50 ms. Here, we investigate (1) how axonal conduction times of corticogeniculate (CG) neurons are related to the visual information conveyed to the thalamus, and (2) how alert versus nonalert awake brain states affect visual processing across the spectrum of CG conduction times. In awake female Dutch-Belted rabbits, we found 58% of CG neurons to be visually responsive, and 42% to be unresponsive. All responsive CG neurons had simple, orientation-selective receptive fields, and generated sustained responses to stationary stimuli. CG axonal conduction times were strongly related to modulated firing rates (F1 values) generated by drifting grating stimuli, and their associated interspike interval distributions, suggesting a continuum of visual responsiveness spanning the spectrum of axonal conduction times. CG conduction times were also significantly related to visual response latency, contrast sensitivity (C-50 values), directional selectivity, and optimal stimulus velocity. Increasing alertness did not cause visually unresponsive CG neurons to become responsive and did not change the response linearity (F1/F0 ratios) of visually responsive CG neurons. However, for visually responsive CG neurons, increased alertness nearly doubled the modulated response amplitude to optimal visual stimulation (F1 values), significantly shortened response latency, and dramatically increased response reliability. These effects of alertness were uniform across the broad spectrum of CG axonal conduction times. SIGNIFICANCE STATEMENT Corticothalamic neurons of layer 6 send a dense feedback projection to thalamic nuclei that provide input to sensory neocortex. While sensory information reaches the cortex after brief thalamocortical axonal delays, corticothalamic axons can exhibit conduction delays of <2 ms to 40-50 ms. Here, in the corticogeniculate

  2. Fcγ receptor-mediated inflammation inhibits axon regeneration.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

  3. Effects of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis

    DEFF Research Database (Denmark)

    Larsen, Mogens; Røntved, Christine Maria; Theil, Peter Kappel

    2017-01-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at –14, +4, +15, and +29 DRTC by measuring [13C]Phe isotopic...... enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[13C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [125I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [13C...

  4. Protein synthesis in muscle cultures from patients with duchenne muscular dystrophy

    International Nuclear Information System (INIS)

    Ionasescu, V.; Zellweger, H.; Ionasescu, R.; Lara-Braud, C.; Cancilla, P.A.

    1976-01-01

    Muscle samples for cultures were obtained from the quadriceps by open biopsy under local anesthesia in five patients with early stage of Duchenne muscular dystrophy (DMD) and 10 controls. Primary cultures were grown in Eagle's Minimum Essential Medium (MEM) with 20 per cent fetal calf serum. After 4 weeks, cells were trypsinized, counted, subcultured for 5 days in MEM with 5 per cent horse serum and finally incubated for 4 h with ( 3 H) leucine. Total protein synthesis showed a significant decrease (ALF OF CONTROL VALUES) only in muscle cultures from patients with DMD. Addition of calcium chloride alone or with A23187 ionophore normalized this defect in protein synthesis. By contrast, myosin heavy chain synthesis was measured and found normal in all patients. (author)

  5. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    Science.gov (United States)

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

  6. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis

    International Nuclear Information System (INIS)

    Drillien, R.; Spehner, D.; Kirn, A.

    1978-01-01

    Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by uv irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective

  7. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    Science.gov (United States)

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

    Science.gov (United States)

    Lu, Hao; Yuan, Wenqiao; Zhou, Jack; Chong, Parkson Lee-Gau

    2015-09-01

    The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system.

  9. [Effect of metalaxyl on the synthesis of RNA, DNA and protein in Phytophthora nicotianae].

    Science.gov (United States)

    Wollgiehn, R; Bräutigam, E; Schumann, B; Erge, D

    1984-01-01

    Metalaxyl is used to control diseases caused by fungi of the order of the Perenosporales. We investigated the action of this fungicid eon nucleic acid and protein synthesis in liquid cultures of Phytophthora nicotianae. The uptake of 32P, 3H-uridine, 3H-thymidine and 14C-leucine as precursors of nuclei acid and protein synthesis by the mycelium was not inhibited by metalaxyl. RNA synthesis as indicated by 3H-uridine incorporation was strongly inhibited (about 80%) by 0.5 micrograms/ml of metalaxyl. The inhibition was visible already few minutes after addition of the toxicant. Since the inhibition of incorporation of 3H-thymidine into DNA and of 14C-leucine into protein became significant 2-3 hours later, we conclude that metalaxyl primarily interfers with RNA synthesis. Synthesis of ribosomal RNA is more affected (more than 90%) than that of tRNA (about 55%) and poly(A)-containing RNA. Since in the presence of actinomycin, in contrast to metalaxyl, protein synthesis is inhibited immediately as a consequence of complete inhibition of RNA synthesis and of the short life-time of mRNA, it is also evident that mRNA synthesis is less strongly inhibited, at least during the early period of metalaxyl action. The molecular mechanism of metalaxyl inhibition of the transcription process remains open. The fungicide did not inhibit the activity of a partially purified RNA polymerase isolated from the fungus. On the other hand, the RNA synthesis (14C-UTP-incorporation) by a cell homogenate and by isolated nuclear fractions was inhibited significantly. Possibilities of the molecular action of metalaxyl are discussed. The RNA synthesis of some plant systems (cell cultures of Lycopersicon peruvianum, isolated nuclei from the same cell cultures, purified RNA polymerase from Spinacia oleracea chloroplasts) was not inhibited by metalaxyl, not even at high concentrations.

  10. MECHANISMS IN ENDOCRINOLOGY: Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review.

    Science.gov (United States)

    Trommelen, Jorn; Groen, Bart B L; Hamer, Henrike M; de Groot, Lisette C P G M; van Loon, Luc J C

    2015-07-01

    Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. To assess whether exogenous insulin administration increases muscle protein synthesis rates in young and older adults. A systematic review of clinical trials was performed and the presence or absence of an increase in muscle protein synthesis rate was reported for each individual study arm. In a stepwise manner, multiple models were constructed that excluded study arms based on the following conditions: model 1, concurrent hyperaminoacidemia; model 2, insulin-induced hypoaminoacidemia; model 3, supraphysiological insulin concentrations; and model 4, older, more insulin resistant, subjects. From the presented data in the current systematic review, we conclude that: i) exogenous insulin and amino acid administration effectively increase muscle protein synthesis, but this effect is attributed to the hyperaminoacidemia; ii) exogenous insulin administered systemically induces hypoaminoacidemia which obviates any insulin-stimulatory effect on muscle protein synthesis; iii) exogenous insulin resulting in supraphysiological insulin levels exceeding 50, 000  pmol/l may effectively augment muscle protein synthesis; iv) exogenous insulin may have a diminished effect on muscle protein synthesis in older adults due to age-related anabolic resistance; and v) exogenous insulin administered systemically does not increase muscle protein synthesis in healthy, young adults. © 2015 European Society of Endocrinology.

  11. Neurotrophin Signaling via Long-Distance Axonal Transport

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    Chowdary, Praveen D.; Che, Dung L.; Cui, Bianxiao

    2012-05-01

    Neurotrophins are a family of target-derived growth factors that support survival, development, and maintenance of innervating neurons. Owing to the unique architecture of neurons, neurotrophins that act locally on the axonal terminals must convey their signals across the entire axon for subsequent regulation of gene transcription in the cell nucleus. This long-distance retrograde signaling, a motor-driven process that can take hours or days, has been a subject of intense interest. In the last decade, live-cell imaging with high sensitivity has significantly increased our capability to track the transport of neurotrophins, their receptors, and subsequent signals in real time. This review summarizes recent research progress in understanding neurotrophin-receptor interactions at the axonal terminal and their transport dynamics along the axon. We emphasize high-resolution studies at the single-molecule level and also discuss recent technical advances in the field.

  12. Mitochondrial Protein Synthesis, Import, and Assembly

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    Fox, Thomas D.

    2012-01-01

    The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

  13. Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

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    Patricia J Ward

    Full Text Available Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2, we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2 to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555 was greater in mice that received optical treatment. Thus, the acute (1 hour, one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-. We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

  14. DCB-3503, a tylophorine analog, inhibits protein synthesis through a novel mechanism.

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    Ying Wang

    Full Text Available BACKGROUND: DCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line and HepG2 (human hepatocellular cancer cell line tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, beta-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins. CONCLUSION/SIGNIFICANCE: The inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

  15. Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis II. Reversal of the RNase effect of protein synthesis by polymethacrylic acid

    NARCIS (Netherlands)

    Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

    1961-01-01

    The ribonuclease inhibited protein synthesis and respiration of yeast protoplasts can be restored by the addition of several polyanionic compounds, among which polymethacrylic acid proved to be the most effective one. The results of preliminary experiments with the ultracentrifuge indicate a

  16. Axonal inclusions in the crab Hemigrapsus nudus.

    Science.gov (United States)

    Smith, R S

    1978-10-01

    Light microscopic examination of living giant axons from the walking legs of Hemigrapsus nudus revealed intra-axonal inclusions which were usually several tens of micrometers long and about 5 micron wide. The inclusions were filled with small light-scattering particles. The inclusions were shown, by thin section electron microscopy, to be composed largely 68% by volume) of mitochondria. Each inclusion was surrounded by membrane bounded spaces which are presumed to represent a part of the smooth endoplasmic reticulum. Similar inclusions were not found in the leg axons of a variety of other decapod crustaceans.

  17. Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1.

    Science.gov (United States)

    Henninger, Nils; Bouley, James; Sikoglu, Elif M; An, Jiyan; Moore, Constance M; King, Jean A; Bowser, Robert; Freeman, Marc R; Brown, Robert H

    2016-04-01

    Axonal degeneration is a critical, early event in many acute and chronic neurological disorders. It has been consistently observed after traumatic brain injury, but whether axon degeneration is a driver of traumatic brain injury remains unclear. Molecular pathways underlying the pathology of traumatic brain injury have not been defined, and there is no efficacious treatment for traumatic brain injury. Here we show that mice lacking the mouse Toll receptor adaptor Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) gene, a key mediator of Wallerian degeneration, demonstrate multiple improved traumatic brain injury-associated phenotypes after injury in a closed-head mild traumatic brain injury model. Sarm1(-/-) mice developed fewer β-amyloid precursor protein aggregates in axons of the corpus callosum after traumatic brain injury as compared to Sarm1(+/+) mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phophorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after traumatic brain injury. Strikingly, whereas wild-type mice exibited a number of behavioural deficits after traumatic brain injury, we observed a strong, early preservation of neurological function in Sarm1(-/-) animals. Finally, using in vivo proton magnetic resonance spectroscopy we found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1(-/-) mice compared to controls immediately following traumatic brain injury. Our results indicate that the SARM1-mediated prodegenerative pathway promotes pathogenesis in traumatic brain injury and suggest that anti-SARM1 therapeutics are a viable approach for preserving neurological function after traumatic brain injury. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Con-nectin axons and dendrites.

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    Beaudoin, Gerard M J

    2006-07-03

    Unlike adherens junctions, synapses are asymmetric connections, usually between axons and dendrites, that rely on various cell adhesion molecules for structural stability and function. Two cell types of adhesion molecules found at adherens junctions, cadherins and nectins, are thought to mediate homophilic interaction between neighboring cells. In this issue, Togashi et al. (see p. 141) demonstrate that the differential localization of two heterophilic interacting nectins mediates the selective attraction of axons and dendrites in cooperation with cadherins.

  19. A growing field: The regulation of axonal regeneration by Wnt signaling.

    Science.gov (United States)

    Garcia, Armando L; Udeh, Adanna; Kalahasty, Karthik; Hackam, Abigail S

    2018-01-01

    The canonical Wnt/β-catenin pathway is a highly conserved signaling cascade that plays critical roles during embryogenesis. Wnt ligands regulate axonal extension, growth cone guidance and synaptogenesis throughout the developing central nervous system (CNS). Recently, studies in mammalian and fish model systems have demonstrated that Wnt/β-catenin signaling also promotes axonal regeneration in the adult optic nerve and spinal cord after injury, raising the possibility that Wnt could be developed as a therapeutic strategy. In this review, we summarize experimental evidence that reveals novel roles for Wnt signaling in the injured CNS, and discuss possible mechanisms by which Wnt ligands could overcome molecular barriers inhibiting axonal growth to promote regeneration. A central challenge in the neuroscience field is developing therapeutic strategies that induce robust axonal regeneration. Although adult axons have the capacity to respond to axonal guidance molecules after injury, there are several major obstacles for axonal growth, including extensive neuronal death, glial scars at the injury site, and lack of axonal guidance signals. Research in rodents demonstrated that activation of Wnt/β-catenin signaling in retinal neurons and radial glia induced neuronal survival and axonal growth, but that activation within reactive glia at the injury site promoted proliferation and glial scar formation. Studies in zebrafish spinal cord injury models confirm an axonal regenerative role for Wnt/β-catenin signaling and identified the cell types responsible. Additionally, in vitro and in vivo studies demonstrated that Wnt induces axonal and neurite growth through transcription-dependent effects of its central mediator β-catenin, potentially by inducing regeneration-promoting genes. Canonical Wnt signaling may also function through transcription-independent interactions of β-catenin with cytoskeletal elements, which could stabilize growing axons and control growth cone

  20. The Alzheimer's β-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb

    Directory of Open Access Journals (Sweden)

    Rajapaksha Tharinda W

    2011-12-01

    Full Text Available Abstract Background The β-secretase, β-site amyloid precursor protein cleaving enzyme 1 (BACE1, is a prime therapeutic target for lowering cerebral β-amyloid (Aβ levels in Alzheimer's disease (AD. Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1-/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs, a well-studied model of axon targeting in vivo. Results We bred BACE1-/- mice with gene-targeted mice in which GFP is expressed from the loci of two odorant-receptors (ORs, MOR23 and M72, and olfactory marker protein (OMP to produce offspring that were heterozygous for MOR23-GFP, M72-GFP, or OMP-GFP and were either BACE1+/+ or BACE1-/-. BACE1-/- mice had olfactory bulbs (OBs that were smaller and weighed less than OBs of BACE1+/+ mice. In wild-type mice, BACE1 was present in OSN axon terminals in OB glomeruli. In whole-mount preparations and tissue sections, many OB glomeruli from OMP-GFP; BACE1-/- mice were malformed compared to wild-type glomeruli. MOR23-GFP; BACE1-/- mice had an irregular MOR23 glomerulus that was innervated by randomly oriented, poorly fasciculated OSN axons compared to BACE1+/+ mice. Most importantly, M72-GFP; BACE1-/- mice exhibited M72 OSN axons that were mis-targeted to ectopic glomeruli, indicating impaired axon guidance in BACE1-/- mice. Conclusions Our results demonstrate that BACE1 is required for the accurate targeting of OSN axons and the proper formation of glomeruli in the OB, suggesting a role for BACE1 in

  1. SAD kinases sculpt axonal arbors of sensory neurons through long- and short-term responses to neurotrophin signals.

    Science.gov (United States)

    Lilley, Brendan N; Pan, Y Albert; Sanes, Joshua R

    2013-07-10

    Extrinsic cues activate intrinsic signaling mechanisms to pattern neuronal shape and connectivity. We showed previously that three cytoplasmic Ser/Thr kinases, LKB1, SAD-A, and SAD-B, control early axon-dendrite polarization in forebrain neurons. Here, we assess their role in other neuronal types. We found that all three kinases are dispensable for axon formation outside of the cortex but that SAD kinases are required for formation of central axonal arbors by subsets of sensory neurons. The requirement for SAD kinases is most prominent in NT-3 dependent neurons. SAD kinases transduce NT-3 signals in two ways through distinct pathways. First, sustained NT-3/TrkC signaling increases SAD protein levels. Second, short-duration NT-3/TrkC signals transiently activate SADs by inducing dephosphorylation of C-terminal domains, thereby allowing activating phosphorylation of the kinase domain. We propose that SAD kinases integrate long- and short-duration signals from extrinsic cues to sculpt axon arbors within the CNS. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Two transcription products of the vesicular stomatitis virus genome may control L-cell protein synthesis

    International Nuclear Information System (INIS)

    Dunigan, D.D.; Lucas-Lenard, J.M.

    1983-01-01

    When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate of protein synthesis ranging from 20 to 85% of that in mock-infected cells. Vesicular stomatitis virus, irradiated with increasing doses of UV light, eventually loses this capacity to inhibit protein synthesis. The UV inactivation curve was biphasic, suggesting that transcription of two regions of the viral genome is necessary for the virus to become inactivated in this capacity. The first transcription produced corresponded to about 373 nucleotides, and the second corresponded to about 42 nucleotides. Inhibition of transcription of the larger product by irradiating the virus with low doses of UV light left a residual inhibition of protein synthesis consisting of approximately 60 to 65% of the total inhibition. This residual inhibition could be obviated by irradiating the virus with a UV dose of greater than 20,000 ergs/mm 2 and was thus considered to represent the effect of the smaller transcription product. In the R1 mutant of another author, the inhibition of transcription of the larger product sufficed to restore protein synthesis to the mock-infected level, suggesting that the smaller transcription product is nonfunctional with respect to protein synthesis inhibition. Extracts from cells infected with virus irradiated with low doses of UV light showed a protein synthesis capacity quite similar to that of their in vivo counterparts, indicating that these extracts closely reflect the in vivo effects of virus infection

  3. Mechanisms of Distal Axonal Degeneration in Peripheral Neuropathies

    Science.gov (United States)

    Cashman, Christopher R.; Höke, Ahmet

    2015-01-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wlds) and Sarmknockout animal models. These studies have shown axonal degeneration to occur througha programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  4. Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level

    Science.gov (United States)

    Asante, Curtis O; Wallace, Victoria C; Dickenson, Anthony H

    2009-01-01

    Background The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation whose action can be inhibited by the drug rapamycin. Forms of long-term plasticity require protein synthesis and evidence indicates that mRNA in dendrites, axon terminals and cell bodies is essential for long-term synaptic plasticity. Specific to pain, shifts in pain thresholds and responsiveness are an expression of neuronal plasticity and this likely contributes to persistent pain. We investigated this by inhibiting the activity of mTOR with rapamycin at the spinal level, of rats that were subjected to the formalin test, using both behavioural and electrophysiological techniques. Results For in vivo electrophysiology, Sprague Dawley rats were fully anaesthetised and single-unit extracellular recordings were obtained from lamina V wide dynamic range (WDR) dorsal horn spinal neurones at the region where input is received from the hind paw. Neuronal responses from naive rats showed that rapamycin-sensitive pathways were important in nociceptive-specific C-fibre mediated transmission onto WDR neurones as well mechanically-evoked responses since rapamycin was effective in attenuating these measures. Formalin solution was injected into the hind paw prior to which, rapamycin or vehicle was applied directly onto the exposed spinal cord. When rapamycin was applied to the spinal cord prior to hind paw formalin injection, there was a significant attenuation of the prolonged second phase of the formalin test, which comprises continuing afferent input to the spinal cord, neuronal hyperexcitability and an activated descending facilitatory drive from the brainstem acting on spinal neurones. In accordance with electrophysiological data, behavioural studies showed that rapamycin attenuated behavioural hypersensitivity elicited by formalin injection into the hind paw. Conclusion We conclude that mTOR has a role in maintaining persistent pain states via mRNA translation and thus protein

  5. Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level

    Directory of Open Access Journals (Sweden)

    Wallace Victoria C

    2009-06-01

    Full Text Available Abstract Background The mammalian target of rapamycin (mTOR is a key regulator of mRNA translation whose action can be inhibited by the drug rapamycin. Forms of long-term plasticity require protein synthesis and evidence indicates that mRNA in dendrites, axon terminals and cell bodies is essential for long-term synaptic plasticity. Specific to pain, shifts in pain thresholds and responsiveness are an expression of neuronal plasticity and this likely contributes to persistent pain. We investigated this by inhibiting the activity of mTOR with rapamycin at the spinal level, of rats that were subjected to the formalin test, using both behavioural and electrophysiological techniques. Results For in vivo electrophysiology, Sprague Dawley rats were fully anaesthetised and single-unit extracellular recordings were obtained from lamina V wide dynamic range (WDR dorsal horn spinal neurones at the region where input is received from the hind paw. Neuronal responses from naive rats showed that rapamycin-sensitive pathways were important in nociceptive-specific C-fibre mediated transmission onto WDR neurones as well mechanically-evoked responses since rapamycin was effective in attenuating these measures. Formalin solution was injected into the hind paw prior to which, rapamycin or vehicle was applied directly onto the exposed spinal cord. When rapamycin was applied to the spinal cord prior to hind paw formalin injection, there was a significant attenuation of the prolonged second phase of the formalin test, which comprises continuing afferent input to the spinal cord, neuronal hyperexcitability and an activated descending facilitatory drive from the brainstem acting on spinal neurones. In accordance with electrophysiological data, behavioural studies showed that rapamycin attenuated behavioural hypersensitivity elicited by formalin injection into the hind paw. Conclusion We conclude that mTOR has a role in maintaining persistent pain states via m

  6. Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

    Science.gov (United States)

    Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

    1998-01-01

    In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

  7. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-01-01

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [ 3 H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [ 3 H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  8. Sigma-1 receptor agonist increases axon outgrowth of hippocampal neurons via voltage-gated calcium ions channels.

    Science.gov (United States)

    Li, Dong; Zhang, Shu-Zhuo; Yao, Yu-Hong; Xiang, Yun; Ma, Xiao-Yun; Wei, Xiao-Li; Yan, Hai-Tao; Liu, Xiao-Yan

    2017-12-01

    Sigma-1 receptors (Sig-1Rs) are unique endoplasmic reticulum proteins that have been implicated in both neurodegenerative and ischemic diseases, such as Alzheimer's disease and stroke. Accumulating evidence has suggested that Sig-1R plays a role in neuroprotection and axon outgrowth. The underlying mechanisms of Sig-1R-mediated neuroprotection have been well elucidated. However, the mechanisms underlying the effects of Sig-1R on axon outgrowth are not fully understood. To clarify this issue, we utilized immunofluorescence to compare the axon lengths of cultured naïve hippocampal neurons before and after the application of the Sig-1R agonist, SA4503. Then, electrophysiology and immunofluorescence were used to examine voltage-gated calcium ion channel (VGCCs) currents in the cell membranes and growth cones. We found that Sig-1R activation dramatically enhanced the axonal length of the naïve hippocampal neurons. Application of the Sig-1R antagonist NE100 and gene knockdown techniques both demonstrated the effects of Sig-1R. The growth-promoting effect of SA4503 was accompanied by the inhibition of voltage-gated Ca 2+ influx and was recapitulated by incubating the neurons with the L-type, N-type, and P/Q-type VGCC blockers, nimodipine, MVIIA and ω-agatoxin IVA, respectively. This effect was unrelated to glial cells. The application of SA4503 transformed the growth cone morphologies from complicated to simple, which favored axon outgrowth. Sig-1R activation can enhance axon outgrowth and may have a substantial influence on neurogenesis and neurodegenerative diseases. © 2017 John Wiley & Sons Ltd.

  9. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Proteomic analysis in giant axonal neuropathy: new insights into disease mechanisms.

    Science.gov (United States)

    Mussche, Silke; De Paepe, Boel; Smet, Joél; Devreese, Katrien; Lissens, Willy; Rasic, Vedrana Milic; Murnane, Matthew; Devreese, Bart; Van Coster, Rudy

    2012-08-01

    Giant axonal neuropathy (GAN) is a progressive hereditary disease that affects the peripheral and central nervous systems. It is characterized morphologically by aggregates of intermediate filaments in different tissues. Mutations have been reported in the gene that codes for gigaxonin. Nevertheless, the underlying molecular mechanism remains obscure. Cell lines from 4 GAN patients and 4 controls were analyzed by iTRAQ. Among the dysregulated proteins were ribosomal protein L29, ribosomal protein L37, galectin-1, glia-derived nexin, and aminopeptidase N. Also, nuclear proteins linked to formin-binding proteins were found to be dysregulated. Although the major role of gigaxonin is reported to be degradation of cytoskeleton-associated proteins, the amount of 76 structural cytoskeletal proteins was unaltered. Several of the dysregulated proteins play a role in cytoskeletal reorganization. Based on these findings, we speculate that disturbed cytoskeletal regulation is responsible for formation of aggregates of intermediate filaments. Copyright © 2012 Wiley Periodicals, Inc.

  11. The cholinergic ligand binding material of axonal membranes

    International Nuclear Information System (INIS)

    Mautner, H.G.; Coronado, R.; Jumblatt, J.E.

    1986-01-01

    Choline acetyltransferase and acetylcholinesterase, the enzymes responsible for the synthesis and hydrolysis of ACh, are present in nerve fibers. In crustacean peripheral nerves, release of ACh from cut nerve fibers has been demonstrated. Previously closed membrane vesicles have been prepared from lobster walking leg nerve plasma membrane and saturable binding of cholinergic agonsist and antagonists to such membranes have been demonstrated. This paper studies this axonal cholinergic binding material, and elucidates its functions. The binding of tritium-nicotine to lobster nerve plasma membranes was antagonized by a series of cholinergic ligands as well as by a series of local anesthetics. This preparation was capable of binding I 125-alpha-bungarotoxin, a ligand widely believed to be a specific label for nicotinic ACh receptor. The labelling of 50 K petide band with tritium-MBTA following disulfide reduction is illustrated

  12. Ammonium-induced impairment of axonal growth is prevented through glial creatine.

    OpenAIRE

    Braissant, O.; Henry, H.; Villard, A.M.; Zurich, M.G.; Loup, M.; Eilers, B.; Parlascino, G.; Matter, E.; Boulat, O.; Honegger, P.; Bachmann, C.

    2002-01-01

    Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cot...

  13. Axonal transport and secretion of fibrillar forms of α-synuclein, Aβ42 peptide and HTTExon 1.

    Science.gov (United States)

    Brahic, Michel; Bousset, Luc; Bieri, Gregor; Melki, Ronald; Gitler, Aaron D

    2016-04-01

    Accruing evidence suggests that prion-like behavior of fibrillar forms of α-synuclein, β-amyloid peptide and mutant huntingtin are responsible for the spread of the lesions that characterize Parkinson disease, Alzheimer disease and Huntington disease, respectively. It is unknown whether these distinct protein assemblies are transported within and between neurons by similar or distinct mechanisms. It is also unclear if neuronal death or injury is required for neuron-to-neuron transfer. To address these questions, we used mouse primary cortical neurons grown in microfluidic devices to measure the amounts of α-synuclein, Aβ42 and HTTExon1 fibrils transported by axons in both directions (anterograde and retrograde), as well as to examine the mechanism of their release from axons after anterograde transport. We observed that the three fibrils were transported in both anterograde and retrograde directions but with strikingly different efficiencies. The amount of Aβ42 fibrils transported was ten times higher than that of the other two fibrils. HTTExon1 was efficiently transported in the retrograde direction but only marginally in the anterograde direction. Finally, using neurons from two distinct mutant mouse strains whose axons are highly resistant to neurodegeneration (Wld(S) and Sarm1(-/-)), we found that the three different fibrils were secreted by axons after anterograde transport, in the absence of axonal lysis, indicating that trans-neuronal spread can occur in intact healthy neurons. In summary, fibrils of α-synuclein, Aβ42 and HTTExon1 are all transported in axons but in directions and amounts that are specific of each fibril. After anterograde transport, the three fibrils were secreted in the medium in the absence of axon lysis. Continuous secretion could play an important role in the spread of pathology between neurons but may be amenable to pharmacological intervention.

  14. Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

    Directory of Open Access Journals (Sweden)

    Nikki A McLean

    Full Text Available Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.

  15. Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

    Science.gov (United States)

    McLean, Nikki A; Popescu, Bogdan F; Gordon, Tessa; Zochodne, Douglas W; Verge, Valerie M K

    2014-01-01

    Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.

  16. Control of protein synthesis in cell-free extracts of sea urchin embryos

    International Nuclear Information System (INIS)

    Hansen, L.J.; Huang, W.I.; Jagus, R.

    1986-01-01

    Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. 35 S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors

  17. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

  18. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    OpenAIRE

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memo...

  19. Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

    Science.gov (United States)

    Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

    2013-05-30

    Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  20. 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.; Moustacchi, E.

    1975-01-01

    The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phase haploid yeast cells was examined. This was carried out using cycloheximide, a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid-holding of cells at both stages, as well as for the ''petite'' recovery seen after the liquid-holding of exponential phase cells. The characteristic negative liquid-holding effect observed for the UV induction of ''petites'' in stationary phase cells (increase of the frequency of ''petites'' during storage) remained, following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark-holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of protein synthesis

  1. Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

    Science.gov (United States)

    Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

    2018-06-01

    Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24

  2. A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

    Science.gov (United States)

    Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

    2017-11-01

    Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  3. T-Stimulator effect on cotton protein composition and synthesis in salinization stress

    International Nuclear Information System (INIS)

    Ibragimova, E.A.; Nazirova, E.R.; Samarkhodjaeva, N.R.; Nalbandyan, A.A.; Babaev, T.A.

    2004-01-01

    Full text: T-stimulator was established to possess a wide spectrum of physiological effects, to enhance plant adaptation to thermal stress and to increase plant resistance to pathogens. Plant adaptation to unfavorable conditions manifests in changes in many links of metabolism, that of proteins included. We studied effect of cottonseed treatment with T-stimulator on composition and synthesis of plasma membrane proteins upon chloride salinization by means of the radioisotope method. Electrophoretic fractionation of cottonseed plasma membrane proteins showed absence of more than 40 polypeptides with molecular mass from 10 to more than 100 kDa in the cotton root membranes. Major fractions-polypeptides with molecular mass of 61, 53, 46, 25, 21, 20 and 18 kDa constitute about 50% of the total polypeptide composition. The salinization significantly affects the total membrane protein output, proportion of some polypeptides and their synthesis rate. Analysis of phoreogram radioautographs showed that 2-hour exposition of cotton roots to 35 S methionine suppresses synthesis of major polypeptides with molecular mass of 63, 61 and 53 kDa, that of low molecular polypeptides (46, 20, 18 kDa) increasing. Changes in the proportion of major polypeptides in cotton plasma membranes, reduction in rate of biosynthesis of high molecular fractions with the general suppression of label inclusion in the membrane fraction are the evidence for a disturbance in biosynthesis of some membrane proteins in cotton tissue cells upon salinization. The inhibiting effect of salinization on the protein-synthesizing system was observed in plants treated with T-stimulator, but the rate of synthesis in plasma membranes of the treated plants was found significantly higher. The activation of some plasma membrane proteins under T-stimulator effect suggests an association with the increase in adaptation of the treated plants to the disturbing effect of salinization

  4. Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina

    Energy Technology Data Exchange (ETDEWEB)

    Rai, Nagendra Kumar; Ashok, Anushruti [Academy of Scientific and Innovative Research (India); Developmental Toxicology, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR) (India); Rai, Asit; Tripathi, Sachin [Developmental Toxicology, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR) (India); Nagar, Geet Kumar [Endocrinology, CSIR-Central Drug Research Institute (CSIR-CDRI) (India); Mitra, Kalyan [Electron Microscopy Unit, CSIR-CDRI, Lucknow 226001 (India); Bandyopadhyay, Sanghamitra, E-mail: sanghmitra@iitr.res.in [Academy of Scientific and Innovative Research (India); Developmental Toxicology, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR) (India)

    2013-12-01

    Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2′-, 3′-cyclic-nucleotide-3′-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developing rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology. - Highlights: • As, Cd and Pb-mixture, at human relevant dose, demyelinate developing rat CNS. • The attenuation in myelin and axon is synergistic. • The optic nerve and brain demonstrate reduced glutamine synthetase.

  5. Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina

    International Nuclear Information System (INIS)

    Rai, Nagendra Kumar; Ashok, Anushruti; Rai, Asit; Tripathi, Sachin; Nagar, Geet Kumar; Mitra, Kalyan; Bandyopadhyay, Sanghamitra

    2013-01-01

    Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2′-, 3′-cyclic-nucleotide-3′-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developing rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology. - Highlights: • As, Cd and Pb-mixture, at human relevant dose, demyelinate developing rat CNS. • The attenuation in myelin and axon is synergistic. • The optic nerve and brain demonstrate reduced glutamine synthetase.

  6. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

    Directory of Open Access Journals (Sweden)

    Proud Christopher G

    2002-05-01

    Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

  7. Time course of ongoing activity during neuritis and following axonal transport disruption.

    Science.gov (United States)

    Satkeviciute, Ieva; Goodwin, George; Bove, Geoffrey M; Dilley, Andrew

    2018-05-01

    Local nerve inflammation (neuritis) leads to ongoing activity and axonal mechanical sensitivity (AMS) along intact nociceptor axons and disrupts axonal transport. This phenomenon forms the most feasible cause of radiating pain, such as sciatica. We have previously shown that axonal transport disruption without inflammation or degeneration also leads to AMS but does not cause ongoing activity at the time point when AMS occurs, despite causing cutaneous hypersensitivity. However, there have been no systematic studies of ongoing activity during neuritis or noninflammatory axonal transport disruption. In this study, we present the time course of ongoing activity from primary sensory neurons following neuritis and vinblastine-induced axonal transport disruption. Whereas 24% of C/slow Aδ-fiber neurons had ongoing activity during neuritis, few (disruption of axonal transport without inflammation does not lead to ongoing activity in sensory neurons, including nociceptors, but does cause a rapid and transient development of AMS. Because it is proposed that AMS underlies mechanically induced radiating pain, and a transient disruption of axonal transport (as previously reported) leads to transient AMS, it follows that processes that disrupt axonal transport, such as neuritis, must persist to maintain AMS and the associated symptoms. NEW & NOTEWORTHY Many patients with radiating pain lack signs of nerve injury on clinical examination but may have neuritis, which disrupts axonal transport. We have shown that axonal transport disruption does not induce ongoing activity in primary sensory neurons but does cause transient axonal mechanical sensitivity. The present data complete a profile of key axonal sensitivities following axonal transport disruption. Collectively, this profile supports that an active peripheral process is necessary for maintained axonal sensitivities.

  8. Epigenetic regulation of axon and dendrite growth

    Directory of Open Access Journals (Sweden)

    Ephraim F Trakhtenberg

    2012-03-01

    Full Text Available Neuroregenerative therapies for central nervous system (CNS injury, neurodegenerative disease, or stroke require axons of damaged neurons to grow and reinnervate their targets. However, mature mammalian CNS neurons do not regenerate their axons, limiting recovery in these diseases (Yiu and He, 2006. CNS’ regenerative failure may be attributable to the development of an inhibitory CNS environment by glial-associated inhibitory molecules (Yiu and He, 2006, and by various cell-autonomous factors (Sun and He, 2010. Intrinsic axon growth ability also declines developmentally (Li et al., 1995; Goldberg et al., 2002; Bouslama-Oueghlani et al., 2003; Blackmore and Letourneau, 2006 and is dependent on transcription (Moore et al., 2009. Although neurons’ intrinsic capacity for axon growth may depend in part on the panoply of expressed transcription factors (Moore and Goldberg, 2011, epigenetic factors such as the accessibility of DNA and organization of chromatin are required for downstream genes to be transcribed. Thus a potential approach to overcoming regenerative failure focuses on the epigenetic mechanisms regulating regenerative gene expression in the CNS. Here we review molecular mechanisms regulating the epigenetic state of DNA through chromatin modifications, their implications for regulating axon and dendrite growth, and important new directions for this field of study.

  9. Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport

    Science.gov (United States)

    Freundt, Eric C.; Maynard, Nate; Clancy, Eileen K.; Roy, Shyamali; Bousset, Luc; Sourigues, Yannick; Covert, Markus; Melki, Ronald; Kirkegaard, Karla; Brahic, Michel

    2012-01-01

    Objective The lesions of Parkinson's disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. Methods We grew primary cortical mouse neurons in microfluidic devices to separate soma from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. Results Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. Interpretation These results support the hypothesis that the progression of Parkinson's disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be transsynaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extra-cellular phase of their journey. PMID:23109146

  10. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

    Science.gov (United States)

    Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

    2008-10-01

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

  11. Axon density and axon orientation dispersion in children born preterm

    NARCIS (Netherlands)

    Kelly, Claire E.; Thompson, Deanne K.; Chen, Jian; Leemans, Alexander; Adamson, Christopher L.; Inder, Terrie E.; Cheong, Jeanie L Y; Doyle, Lex W.; Anderson, Peter J.

    2016-01-01

    Background Very preterm birth (VPT, <32 weeks' gestation) is associated with altered white matter fractional anisotropy (FA), the biological basis of which is uncertain but may relate to changes in axon density and/or dispersion, which can be measured using Neurite Orientation Dispersion and Density

  12. Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

    Science.gov (United States)

    Fernie, Alisdair R.; Stitt, Mark

    2015-01-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

  13. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

    Bornø, Andreas; Hulston, Carl J; van Hall, Gerrit

    2014-01-01

    In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically...

  14. Sulfur in human nutrition - effects beyond protein synthesis

    NARCIS (Netherlands)

    Gertjan Schaafsma

    2008-01-01

    That sulfur is essential to humans is based on the requirement of S-animo acids for normal growth and maintenance of nitrogen balance and not on the optimization of metabolic proccesses involving the synthesis of non-protein sulphur containing compounds. This paper reviews the significance of sulfur

  15. Long-term olfactory memories are stabilised via protein synthesis in Camponotus fellah ants

    DEFF Research Database (Denmark)

    Guerrieri, Fernando Javier; D'Ettorre, Patrizia; Deveaud, J-M.

    2011-01-01

    -chain hydrocarbons, one paired with sucrose and the other with quinine solution. Differential conditioning leads to the formation of a long-term memory retrievable at least 72¿h after training. Long-term memory consolidation was impaired by the ingestion of cycloheximide, a protein synthesis blocker, prior...... to conditioning. Cycloheximide did not impair acquisition of either short-term memory (10¿min) or early and late mid-term memories (1 or 12¿h). These results show that, upon olfactory learning, ants form different memories with variable molecular bases. While short- and mid-term memories do not require protein...... synthesis, long-term memories are stabilised via protein synthesis. Our behavioural protocol opens interesting research avenues to explore the cellular and molecular bases of olfactory learning and memory in ants....

  16. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Directory of Open Access Journals (Sweden)

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  17. Effect of acute maternal starvation on tyrosine metabolism and protein synthesis in fetal sheep

    International Nuclear Information System (INIS)

    Krishnamurti, C.R.; Schaefer, A.L.

    1984-01-01

    To determine the effects of acute maternal starvation on intrauterine growth, tyrosine concentration and specific activity values in plasma, intracellular free and protein bound pools were determined in catheterized ovine fetuses following an 8 h continuous infusion of L-[2,3,5,6 3 H] or L-[U- 14 C] tyrosine into the ewe and fetus respectively at 115-125 days of gestation. From the kinetic data the rates of whole body and tissue fractional protein synthesis were calculated. Although placental protein synthesis was not significantly changed as a result of acute maternal starvation, fetal whole body protein synthesis was reduced from 63 g/d/kg in the fed to 25 g/d/kg in the starved condition. There was also a 10 fold reduction in the net placental transfer of tyrosine to the fetus in the starved ewes. In addition, a three fold increase was observed in the quantity of tyrosine used for oxidation by the fetuses of starved ewes, changing from 5.2% of tyrosine net utilization in the fed to 13.7% in the starved condition. Significant reductions in tissue fractional protein synthesis rates were also seen in the liver, brain, lung kidney and GIT tissues from 78, 37, 65, 45 and 71%/d respectively in the fed to 12, 10, 23, 22 and 35%/d in the fetuses of starved ewes. The data indicate that during acute maternal starvation the sheep fetus utilizes more tyrosine for oxidation and less for anabolic purposes which is reflected in a decrease both in whole body and tissue fractional rates of protein synthesis

  18. Highly effective photonic cue for repulsive axonal guidance.

    Directory of Open Access Journals (Sweden)

    Bryan J Black

    Full Text Available In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods. These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm.

  19. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    Science.gov (United States)

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Axon diameter mapping in crossing fibers with diffusion MRI

    DEFF Research Database (Denmark)

    Zhang, Hui; Dyrby, Tim B; Alexander, Daniel C

    2011-01-01

    This paper proposes a technique for a previously unaddressed problem, namely, mapping axon diameter in crossing fiber regions, using diffusion MRI. Direct measurement of tissue microstructure of this kind using diffusion MRI offers a new class of biomarkers that give more specific information about...... tissue than measures derived from diffusion tensor imaging. Most existing techniques for axon diameter mapping assume a single axon orientation in the tissue model, which limits their application to only the most coherently oriented brain white matter, such as the corpus callosum, where the single...... model to enable axon diameter mapping in voxels with crossing fibers. We show in simulation that the technique can provide robust axon diameter estimates in a two-fiber crossing with the crossing angle as small as 45 degrees. Using ex vivo imaging data, we further demonstrate the feasibility...

  1. Radioautographic study of protein synthesis during early embryogenesis of Leptimotarsa decemlineata Say (Coleoptera)

    International Nuclear Information System (INIS)

    Maisonhaute, Claude

    1976-01-01

    Protein synthesis in early embryonic stages of the Colorado beetle has been investigated by radioautography. Radioactive precursor (L. Leucine-3 H) was injected in eggs. At the stage of blastoderm formation amino-acid incorporation decreases sharply: at late blastula stage, incorporation reaches the same levels as during early cleavage, and at gastrula stage becomes higher. Nuclear protein synthesis is first detected during blastoderm formation and increases at gastrula stage [fr

  2. Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells

    International Nuclear Information System (INIS)

    Shaw, J.E.; Epp, C.; Pearson, M.L.; Reeve, J.N.

    1987-01-01

    The patterns of bacteriophage lambda proteins synthesized in UV-irradiated Escherichia coli cells and in anucleate minicells are significantly different; both systems exhibit aberrations of regulation in lambda gene expression. In unirradiated cells or cells irradiated with low UV doses (less than 600 J/m2), regulation of lambda protein synthesis is controlled by the regulatory proteins CI, N, CII, CIII, Cro, and Q. As the UV dose increases, activation of transcription of the cI, rexA, and int genes by CII and CIII proteins fails to occur and early protein synthesis, normally inhibited by the action of Cro, continues. After high UV doses (greater than 2000 J/m2), late lambda protein synthesis does not occur. Progression through the sequence of regulatory steps in lambda gene expression is slower in infected minicells. In minicells, there is no detectable cII- and cIII-dependent synthesis of CI, RexA, or Int proteins and inhibition of early protein synthesis by Cro activity is always incomplete. The synthesis of early b region proteins is not subject to control by CI, N, or Cro proteins, and evidence is presented suggesting that, in minicells, transcription of the early b region is initiated at a promoter(s) within the b region. Proteolytic cleavage of the regulatory proteins O and N and of the capsid proteins C, B, and Nu3 is much reduced in infected minicells. Exposure of minicells to very high UV doses before infection does not completely inhibit late lambda protein synthesis

  3. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

    DEFF Research Database (Denmark)

    Miller, Benjamin F; Olesen, Jens L; Hansen, Mette

    2005-01-01

    We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied...... collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage...... of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise...

  4. Nuclear transport factor directs localization of protein synthesis during mitosis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Meinema, Anne C.; Krasnikov, Viktor; Veenhoff, Liesbeth M.; Poolman, Bert

    Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the

  5. Cooperative Interactions between 480 kDa Ankyrin-G and EB proteins assemble the axon initial segment

    NARCIS (Netherlands)

    Fréal, Amélie|info:eu-repo/dai/nl/412516721; Fassier, Coralie; Le Bras, Barbara; Bullier, Erika; de Gois, Stéphanie; Hazan, Jamilé; Hoogenraad, Casper C.|info:eu-repo/dai/nl/227263502; Couraud, François

    2016-01-01

    The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding

  6. The differential role of cortical protein synthesis in taste memory formation and persistence

    Science.gov (United States)

    Levitan, David; Gal-Ben-Ari, Shunit; Heise, Christopher; Rosenberg, Tali; Elkobi, Alina; Inberg, Sharon; Sala, Carlo; Rosenblum, Kobi

    2016-05-01

    The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola (n⩾5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation (P=8.9E-5), but had no effect on LTM persistence when infused 3 days post acquisition (P=0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced (P=0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an N-methyl-D-aspartate receptor antagonist (P=0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.

  7. Whey and casein labelled with L-[1-13C]-leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2011-01-01

    to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass......), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments......, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after...

  8. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

    International Nuclear Information System (INIS)

    Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

    1986-01-01

    Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of α-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as α-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured

  9. Problem-Solving Test: The Mechanism of Protein Synthesis

    Science.gov (United States)

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and C-terminus, hemoglobin, [alpha]- and [beta]-globin chains, radioactive labeling, [[to the third power]H] and [[to the fourteenth power]C]leucine, cytosol, differential centrifugation, density…

  10. SnoN facilitates axonal regeneration after spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Jiun L Do

    Full Text Available Adult CNS neurons exhibit a reduced capacity for growth compared to developing neurons, due in part to downregulation of growth-associated genes as development is completed. We tested the hypothesis that SnoN, an embryonically regulated transcription factor that specifies growth of the axonal compartment, can enhance growth in injured adult neurons. In vitro, SnoN overexpression in dissociated adult DRG neuronal cultures significantly enhanced neurite outgrowth. Moreover, TGF-β1, a negative regulator of SnoN, inhibited neurite outgrowth, and SnoN over-expression overcame this inhibition. We then examined whether SnoN influenced axonal regeneration in vivo: indeed, expression of a mutant form of SnoN resistant to degradation significantly enhanced axonal regeneration following cervical spinal cord injury, despite peri-lesional upregulation of TGF-β1. Thus, a developmental mechanism that specifies extension of the axonal compartment also promotes axonal regeneration after adult CNS injury.

  11. Viral protein synthesis in cowpea mosaic virus infected protoplasts

    International Nuclear Information System (INIS)

    Rottier, P.

    1980-01-01

    Some aspects of cowpea mosaic virus (CPMV) multiplication in cowpea mesophyll protoplasts were studied. The detection and characterization of proteins whose synthesis is induced or is stimulated upon virus infection was performed with the aid of radioactive labelling. (Auth.)

  12. Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

    OpenAIRE

    E. Ruchti; P.J. Roach; A.A. DePaoli-Roach; P.J. Magistretti; I. Allaman

    2016-01-01

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the ...

  13. Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia

    Science.gov (United States)

    Denton, Kyle R.; Lei, Ling; Grenier, Jeremy; Rodionov, Vladimir; Blackstone, Craig; Li, Xue-Jun

    2013-01-01

    Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. PMID:24123785

  14. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    Science.gov (United States)

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

  15. The limits of adaptation of functional protein synthesis to severe undernutrition

    International Nuclear Information System (INIS)

    Forrester, T.; Jahoor, F.; Reeds, P.

    1996-01-01

    This project was designed to investigate the limits of adaptation of protein metabolism in the stree of severe childhood malnutrition, representing as it does chronic dietary insufficiency of macronutrients and superimposed infection. The tasks included measurement of concentrations and rates of synthesis of nutrient transport proteins and hepatic acute phase proteins inseverely malnourished children during their acute illness and a recovery

  16. Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process.

    Science.gov (United States)

    Jeyapalan, Asumthia S; Orellana, Renan A; Suryawan, Agus; O'Connor, Pamela M J; Nguyen, Hanh V; Escobar, Jeffery; Frank, Jason W; Davis, Teresa A

    2007-08-01

    Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.

  17. Mdivi-1 inhibits astrocyte activation and astroglial scar formation and enhances axonal regeneration after spinal cord injury in rats

    Directory of Open Access Journals (Sweden)

    gang li

    2016-10-01

    Full Text Available After spinal cord injury (SCI, astrocytes become hypertrophic and proliferative, forming a dense network of astroglial processes at the site of the lesion. This constitutes a physical and biochemical barrier to axonal regeneration. Mitochondrial fission regulates cell cycle progression; inhibiting the cell cycle of astrocytes can reduce expression levels of axon growth-inhibitory molecules as well as astroglial scar formation after SCI. We therefore investigated how an inhibitor of mitochondrial fission, Mdivi-1, would affect astrocyte proliferation, astroglial scar formation, and axonal regeneration following SCI in rats. Western blot and immunofluorescent double-labeling showed that Mdivi-1 markedly reduced the expression of the astrocyte marker glial fibrillary acidic protein (GFAP, and a cell proliferation marker, proliferating cell nuclear antigen, in astrocytes 3 days after SCI. Moreover, Mdivi-1 decreased the expression of GFAP and neurocan, a chondroitin sulfate proteoglycan. Notably, immunofluorescent labeling and Nissl staining showed that Mdivi-1 elevated the production of growth-associated protein-43 and increased neuronal survival at 4 weeks after SCI. Finally, hematoxylin-eosin staining and behavioral evaluation of motor function indicated that Mdivi-1 also reduced cavity formation and improved motor function 4 weeks after SCI. Our results confirm that Mdivi-1 promotes motor function after SCI, and indicate that inhibiting mitochondrial fission using Mdivi-1 can inhibit astrocyte activation and astroglial scar formation and contribute to axonal regeneration after SCI in rats.

  18. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

  19. Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals.

    Directory of Open Access Journals (Sweden)

    Yulin Dong

    Full Text Available It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG or FG/tetramethylrhodamine-dextran amine (TMR-DA were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA was injected into the caudal spinal trigeminal nucleus (Vc. The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt, dorsal and ventral medullary reticular formation (MdD, MdV, supratrigeminal nucleus (Vsup and parabrachial nucleus (PBN with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.

  20. Long-term memory for instrumental responses does not undergo protein synthesis-dependent reconsolidation upon retrieval.

    Science.gov (United States)

    Hernandez, Pepe J; Kelley, Ann E

    2004-01-01

    Recent evidence indicates that certain forms of memory, upon recall, may return to a labile state requiring the synthesis of new proteins in order to preserve or reconsolidate the original memory trace. While the initial consolidation of "instrumental memories" has been shown to require de novo protein synthesis in the nucleus accumbens, it is not known whether memories of this type undergo protein synthesis-dependent reconsolidation. Here we show that low doses of the protein synthesis inhibitor anisomycin (ANI; 5 or 20 mg/kg) administered systemically in rats immediately after recall of a lever-pressing task potently impaired performance on the following daily test sessions. We determined that the nature of this impairment was attributable to conditioned taste aversion (CTA) to the sugar reinforcer used in the task rather than to mnemonic or motoric impairments. However, by substituting a novel flavored reinforcer (chocolate pellets) prior to the administration of doses of ANI (150 or 210 mg/kg) previously shown to cause amnesia, a strong CTA to chocolate was induced sparing any aversion to sugar. Importantly, when sugar was reintroduced on the following session, we found that memory for the task was not significantly affected by ANI. Thus, these data suggest that memory for a well-learned instrumental response does not require protein synthesis-dependent reconsolidation as a means of long-term maintenance.

  1. N-docosahexaenoylethanolamine regulates Hedgehog signaling and promotes growth of cortical axons

    Directory of Open Access Journals (Sweden)

    Giorgi Kharebava

    2015-12-01

    Full Text Available Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3 that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine, an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1 transcription and sonic hedgehog (Shh target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO agonist (SAG or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway.

  2. Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

    Science.gov (United States)

    Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R; Alsner, Jan

    2015-01-01

    The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo

  3. Gamma-ray-induced changes in the synthesis of tomato pericarp protein

    International Nuclear Information System (INIS)

    Ferullo, J.M.; Nespoulous, L.; Triantaphylides, C.

    1994-01-01

    The application of massive doses of gamma rays (1–8 kGy) to mature green cherry-tomato fruits led to a transient fall in pericarp tissue protein metabolism within 6h. A separate 3 kGy treatment resulted in the appearance of certain transcripts and proteins, and a reduction in the abundance of others. At the same dose, protein synthesis regained the control level within 24 h, and in addition a new set of proteins was induced. Gamma-induced proteins (referred to as GIPs) were divided into three groups, depending on the time-course of their induction. Group 1 GIPs were synthesized only during the first few hours following treatment, whereas group 2 GIPs were synthesized for at least 48 h. Group 3 GIPs were progressively induced when the control level of synthesis was restored. These results demonstrated that, despite its deleterious effects on DNA and cell structures, irradiation induced an active response in plant tissue. Comparative experiments suggest that the majority of group 1 GIPs might belong to the heat shock protein family. GIPs might play a role in the protection and repair of cellular structures, or may be implicated in physiological disorders triggered by irradiation. (author)

  4. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    Science.gov (United States)

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  5. Modality-Specific Axonal Regeneration: Towards selective regenerative neural interfaces

    Directory of Open Access Journals (Sweden)

    Parisa eLotfi

    2011-10-01

    Full Text Available Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed submodality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type-specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF and neurotrophin-3 (NT-3, to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5 fold compared to that in saline or NT-3, whereas the number of branches increased 3 fold in the NT-3 channels. These results were confirmed using a 3-D Y-shaped in vitro assay showing that the arm containing NGF was able to entice a 5-fold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a Y-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted towards the sural nerve, while N-52+ large diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces.

  6. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

    Directory of Open Access Journals (Sweden)

    Mathieu Desclaux

    Full Text Available BACKGROUND: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi. METHODS AND FINDINGS: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. CONCLUSIONS: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for

  7. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

    Science.gov (United States)

    Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Gimenez Y Ribotta, Minerva; Privat, Alain; Mallet, Jacques

    2009-07-14

    The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration

  8. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

  9. Localization of mRNA in vertebrate axonal compartments by in situ hybridization.

    Science.gov (United States)

    Sotelo-Silveira, José Roberto; Calliari, Aldo; Kun, Alejandra; Elizondo, Victoria; Canclini, Lucía; Sotelo, José Roberto

    2011-01-01

    The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

  10. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

    International Nuclear Information System (INIS)

    Hill, T.M.; Sinden, R.R.; Sadler, J.R.

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

  11. In vitro estimation of rumen microbial protein synthesis of water buffaloes using 30S as tracer

    International Nuclear Information System (INIS)

    Hendratno, C.; Abidin, Z.; Bahaudin, R.; Sastrapradja, D.

    1977-01-01

    An experiment to study the effect of diet and individual differences of animals on the in vitro estimation of rumen microbial protein synthesis in young female water buffaloes using the technique of inorganic 35 S incorporation, is described. The dietary treatments were four combinations of roughage supplemented with cassava meal. From the value of rate constant for dilution of radioactivity in the sulphide pool and percentage of inorganic 35 S incorporated into microbial protein, it can be concluded that individual differences of animals have no influence on the efficiency of microbial protein synthesis. Feed composition, on the other hand, tends to have some influence on the efficiency of protein synthesis(P3O.15). (author)

  12. An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

    Science.gov (United States)

    Huang, Claire Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel R; Leterrier, Christophe; Rasband, Matthew N

    2017-11-22

    Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1 f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K + channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1 f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier. SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in

  13. Effect of inhibition of protein synthesis on the development of thermotolerance

    International Nuclear Information System (INIS)

    Chang, P.Y.; Blakely, E.A.; Gonzalez-Flores, I.

    1986-01-01

    The authors have chosen to use a temperature-sensitive mutant line, CHO-TSH1, which shuts down protein synthesis at nonpermissive temperatures of 40 0 C and above by the inactivation of its cytoplasmic nonmitochondrial leucyl-transfer RNA (t-RNA) synthetase enzyme. The parent cell line, CHO-SC1, was used as the control for these experiments. Exponentially growing, asynchronous CHO-TSH1 and CHO-SC1 cell populations were treated for times up to 8 hours at 41.5 0 C, 42 0 C, and 42.5 0 C. The wild-type cells showed the development of tolerance to heat killing at 41.5 0 C, 42 0 C, and possibly at 42.5 0 C, although the survival level at which tolerance developed at 42.5 0 C was too low to be statistically significant. The CHO-TSH1 mutant cell showed no tolerance at any of those temperatures. The rate of total protein synthesis was measured in both cell lines in pulse-labeling experiments with 3 H-leucine under the conditions of the experiment. Results indicated that the rate of synthesis dropped precipitously within the initial hour of exposure to 42 0 C and remained low during the 3 hours of 42 0 C treatment. When each cell line was returned to 35 0 C after the 3-hour treatment at 42 0 C, protein synthesis immediately resumed and eventually returned to control levels after 7 hours at 35 0 C

  14. Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

    Science.gov (United States)

    Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

    2018-01-01

    DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

  15. Optogenetically enhanced axon regeneration: motor versus sensory neuron-specific stimulation.

    Science.gov (United States)

    Ward, Patricia J; Clanton, Scott L; English, Arthur W

    2018-02-01

    Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system-specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light-sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2-positive neurons whose axons had regenerated successfully was greater following system-specific optical treatment, with no effect on the number of ChR2-negative neurons (whether motor or sensory neurons). We conclude that acute system-specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration-enhancing effect is likely cell autonomous. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  16. PROTEIN SYNTHESIS GAME’: UTILIZING GAME-BASED APPROACH FOR IMPROVING COMMUNICATIVE SKILLS IN A-LEVELS BIOLOGY CLASS

    Directory of Open Access Journals (Sweden)

    Mohd Adlan Ramly

    2017-12-01

    Full Text Available This experimental paper seeks to elucidate the usage of the card game ‘Protein Synthesis Game’ as a student’s learning tool in studying the Biology topic of protein synthesis during an A-Level course. A total of 24 experimental students in 3 induced groups and 24 controlled students in controlled groups were involved in the experiment which began with a pretest on the topic of Protein Synthesis, followed by the experimentation, and ended with a post-test administered after the incubation period. Results indicate that students have better facilitative communicative engagement in learning protein synthesis when playing the game as compared to studying the topic from a book. The data suggests that such communicative engagement may lead to a successful meaningful learning on the students’ part.

  17. Differential effects of methylmercury on the synthesis of protein species in dorsal root ganglia of the rat

    International Nuclear Information System (INIS)

    Kasama, Hidetaka; Itoh, Kazuo; Omata, Saburo; Sugano, Hiroshi

    1989-01-01

    Dorsal root ganglia from control and methylmercury(MeHg)-treated rats were incubated in vitro with 35 S-methionine and the proteins synthesized were analyzed by two-dimensional electrophoresis. The double labelling method, in which proteins of control dorsal root ganglia labelled in vitro with 3 H-leucine were added to each of the two samples as an internal standard, was used to minimize unavoidable errors arising from the resolving procedure itself. The results obtained showed that the effect of MeHg on the synthesis of proteins in dorsal root ganglia was not uniform for individual protein species in the latent period of MeHg intoxication. Among 200 protein species investigated, 157 showed inhibition of synthesis close to that of the total proteins in the tissue (68% of the control). Among the remaining protein species, 20 showed real stimulation of synthesis, whereas 7 were moderately inhibited and 16 were inhibited more strongly than the total proteins in the tissue. These results suggest that the effect of MeHg on the synthetic rates for protein species in dorsal root ganglia differs with the species, and that unusual elevation or reduction of the synthesis of some protein species caused by MeHg may lead to impairment of normal nerve functions. (orig.)

  18. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    Science.gov (United States)

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  19. Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis

    International Nuclear Information System (INIS)

    Hatch, T.P.; Miceli, M.; Silverman, J.A.

    1985-01-01

    Synthesis of protein by the obligate intracellular parasitic bacteria Chlamydia psittaci (6BC) and Chlamydia trachomatis (serovar L2) isolated from host cells (host-free chlamydiae) was demonstrated for the first time. Incorporation of [ 35 S]methionine and [ 35 S]cysteine into trichloroacetic acid-precipitable material by reticulate bodies of chlamydiae persisted for 2 h and was dependent upon a exogenous source of ATP, an ATP-regenerating system, and potassium or sodium ions. Magnesium ions and amino acids stimulated synthesis; chloramphenicol, rifampin, oligomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a proton ionophore) inhibited incorporation. Ribonucleoside triphosphates (other than ATP) had little stimulatory effect. The optimum pH for host-free synthesis was between 7.0 and 7.5. The molecular weights of proteins synthesized by host-free reticulate bodies closely resembled the molecular weights of proteins synthesized by reticulate bodies in an intracellular environment, and included outer membrane proteins. Elementary bodies of chlamydiae were unable to synthesize protein even when incubated in the presence of 10 mM dithiothreitol, a reducing agent which converted the highly disulfide bond cross-linked major outer membrane protein to monomeric form

  20. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Directory of Open Access Journals (Sweden)

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  1. Protein synthesis evaluation in brain and other organs in human by PET

    International Nuclear Information System (INIS)

    Bustany, P.; Comar, D.

    1985-01-01

    The choice of treatment in diseases of the nervous system cannot be based only on symptomatology, but on a presumed underlying pathological state. These pathological states often involve direct modifications of neuronal metabolism. Two areas of cellular biochemistry can be studied in vivo in humans: 1) glucose or oxygen consumption which is mainly responsible for energy and lipid metabolism. 2) amino acid metabolism, which is involved in protein and neurotransmitter synthesis. Here the authors examine protein synthesis, which is the basis of cellular integrity and tissue structure. Study of protein synthesis (PS) by positron emission tomography (PET) is governed by specific requirements dictated by 1) the metabolic pathways we want to explore (the fate of the tracer directly influences the analysis of the results); 2) The construction and validation of a mathematical model to be applied to the computerized images; and 3) the human pathology being studied. The timing of scanning and the experimental protocol must include in their conception some physiological constraints such as volume of organs, rapidity of biological phenomena, etc. All these steps are detailed in the following paragraphs

  2. Prolonged high frequency electrical stimulation is lethal to motor axons of mice heterozygously deficient for the myelin protein P0 gene

    DEFF Research Database (Denmark)

    Alvarez, Susana; Moldovan, Mihai; Krarup, Christian

    2013-01-01

    demyelinating neuropathy reminiscent of CMT Type 1b. Accumulating evidence suggests that impulse conduction can become lethal to acutely demyelinated central and peripheral axons. Here we investigated the vulnerability of motor axons to long-lasting, high-frequency repetitive stimulation (RS) in P₀+/- mice...... as compared to WT littermates at 7, 12, and 20 months of age. RS was carried out in interrupted trains of 200 Hz trains for 3h. Tibial nerves were stimulated at the ankle while the evoked compound muscle action potentials (CMAPs) and the ascending compound nerve action potentials (CNAPs) were recorded from...... aging and the dysmyelinating disease process may contribute to the susceptibility to activity-induced axonal degeneration. It is possible that in aging mice and in P₀+/- there is inadequate energy-dependent Na(+)/K(+) pumping, as indicated by the reduced post-stimulation hyperpolarization, which may...

  3. Is there any relationship between decreased AgNOR protein synthesis and human hair loss?

    Science.gov (United States)

    Eroz, R; Tasdemir, S; Dogan, H

    2012-11-01

    Argyrophilic nucleolar organizing region associated proteins (AgNORs) play roles in cell proliferation and a variety of diseases. We attempted to determine whether decreased NOR protein synthesis causes human hair loss. We studied 21 healthy males who suffered hair loss on the frontal/vertex portion of the head. Hair root cells from normal and hair loss sites were stained for AgNOR. One hundred nuclei per site were evaluated and the AgNOR number and NORa/TNa proportions of individual cells were determined using a computer program. The cells from normal sites had significantly higher AgNOR counts than those from hair loss sites. Also, the cells from the normal sites had significantly higher NORa/TNa than cells from the hair loss sites. In the normal sites, the cells demonstrated more NOR protein synthesis than cells in hair loss sites. Therefore, decreased NOR protein synthesis appears to be related to hair loss in humans.

  4. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions.

    Science.gov (United States)

    Shaginian, Alex; Whitby, Landon R; Hong, Sukwon; Hwang, Inkyu; Farooqi, Bilal; Searcey, Mark; Chen, Jiandong; Vogt, Peter K; Boger, Dale L

    2009-04-22

    The design and solution-phase synthesis of an alpha-helix mimetic library as an integral component of a small-molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate alpha-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 x 20 x 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i + 4, i + 7 amino acid side chains of an alpha-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long-term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead alpha-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an alpha-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

  5. Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

    Science.gov (United States)

    Manjarín, Rodrigo; Columbus, Daniel A; Suryawan, Agus; Nguyen, Hanh V; Hernandez-García, Adriana D; Hoang, Nguyet-Minh; Fiorotto, Marta L; Davis, Teresa

    2016-01-01

    Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

  6. Axon-somatic back-propagation in detailed models of spinal alpha motoneurons

    Directory of Open Access Journals (Sweden)

    Pietro eBalbi

    2015-02-01

    Full Text Available Antidromic action potentials following distal stimulation of motor axons occasionally fail to invade the soma of alpha motoneurons in spinal cord, due to their passing through regions of high non-uniformity.Morphologically detailed conductance-based models of cat spinal alpha motoneurons have been developed, with the aim to reproduce and clarify some aspects of the electrophysiological behavior of the antidromic axon-somatic spike propagation. Fourteen 3D morphologically detailed somata and dendrites of cat spinal alpha motoneurons have been imported from an open-access web-based database of neuronal morphologies, NeuroMorpho.org, and instantiated in neurocomputational models. An axon hillock, an axonal initial segment and a myelinated axon are added to each model.By sweeping the diameter of the axonal initial segment (AIS and the axon hillock, as well as the maximal conductances of sodium channels at the AIS and at the soma, the developed models are able to show the relationships between different geometric and electrophysiological configurations and the voltage attenuation of the antidromically travelling wave.In particular, a greater than usually admitted sodium conductance at AIS is necessary and sufficient to overcome the dramatic voltage attenuation occurring during antidromic spike propagation both at the myelinated axon-AIS and at the AIS-soma transitions.

  7. A Communication Theoretical Modeling of Axonal Propagation in Hippocampal Pyramidal Neurons.

    Science.gov (United States)

    Ramezani, Hamideh; Akan, Ozgur B

    2017-06-01

    Understanding the fundamentals of communication among neurons, known as neuro-spike communication, leads to reach bio-inspired nanoscale communication paradigms. In this paper, we focus on a part of neuro-spike communication, known as axonal transmission, and propose a realistic model for it. The shape of the spike during axonal transmission varies according to previously applied stimulations to the neuron, and these variations affect the amount of information communicated between neurons. Hence, to reach an accurate model for neuro-spike communication, the memory of axon and its effect on the axonal transmission should be considered, which are not studied in the existing literature. In this paper, we extract the important factors on the memory of axon and define memory states based on these factors. We also describe the transition among these states and the properties of axonal transmission in each of them. Finally, we demonstrate that the proposed model can follow changes in the axonal functionality properly by simulating the proposed model and reporting the root mean square error between simulation results and experimental data.

  8. Protein synthesis in the rat brain: a comparative in vivo and in vitro study in immature and adult animals

    International Nuclear Information System (INIS)

    Shahbazian, F.M.

    1985-01-01

    Rates of protein synthesis of CNS and other organs were compared in immature and adult rats by in vivo and slice techniques with administration of flooding doses of labeled precursor. The relationship between synthesis and brain region, cell type, subcellular fraction, or MW was examined. Incorporation of [ 14 C]valine into protein of CNS regions in vivo was about 1.2% per hour for immature rats and 0.6% for adults. For slices, the rates decreased significantly more in adults. In adult organs, the highest synthesis rate in vivo was found in liver (2.2% per hour) followed by kidney, spleen, lung, heart, brain, and muscle (0.5% per hour). In immature animals synthesis was highest in liver and spleen (2.5% per hour) and lowest in muscle (0.9% per hour). Slices all showed lower rates than in vivo, especially in adults. In vivo, protein synthesis rates of immature neurons and astrocytes and adult neurons exceeded those of whole brain, while that in adult astrocytes was the same. These results demonstrate a developmental difference of protein synthesis (about double in immature animals) in all brain cells, cell fractions and most brain protein. Similarly the decreased synthesis in brain slices - especially in adults, affects most proteins and structural elements

  9. Co-immobilization of semaphorin3A and nerve growth factor to guide and pattern axons.

    Science.gov (United States)

    McCormick, Aleesha M; Jarmusik, Natalie A; Leipzig, Nic D

    2015-12-01

    distribution. The novelty of this research is that we provide a specific attachment method to immobilize an attractive signal, nerve growth factor, along with an inhibitory cue, semaphorin3A, to a substrate in order to analyze the interplay of these proteins on axon guidance responses. The scientific impact of this manuscript is that we show that dual-cued platforms are necessary in order to finetune and tailor specific axon responses for varying neuronal regenerative purposes. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  10. Dendrobium nobile Lindl alkaloid, a novel autophagy inducer, protects against axonal degeneration induced by Aβ25-35 in hippocampus neurons in vitro.

    Science.gov (United States)

    Li, Li-Sheng; Lu, Yan-Liu; Nie, Jing; Xu, Yun-Yan; Zhang, Wei; Yang, Wen-Jin; Gong, Qi-Hai; Lu, Yuan-Fu; Lu, Yang; Shi, Jing-Shan

    2017-04-01

    Axonal degeneration is a pathological symbol in the early stage of Alzheimer's disease (AD), which can be triggered by amyloid-β (Aβ) peptide deposition. Growing evidence indicates that deficit of autophagy eventually leads to the axonal degeneration. Our previous studies have shown that Dendrobium nobile Lindl alkaloid (DNLA) had protective effect on neuron impairment in vivo and in vitro; however, the underlying mechanisms is still unclear. We exposed cultured hippocampus neurons to Aβ 25-35 to investigate the effect of DNLA in vitro. Axonal degeneration was evaluated by immunofluorescence staining and MTT assay. Neurons overexpressing GFP-LC3B were used to measure the formation of autophagosome. Autophagosome-lysosome fusion, the lysosomal pH, and cathepsin activity were assessed to reflect autophagy process. Proteins of interest were analyzed by Western blot. DNLA pretreatment significantly inhibited axonal degeneration induced by Aβ 25-35 peptide in vitro. Further studies revealed DNLA treatment increased autophagic flux through promoting formation and degradation of autophagosome in hippocampus neurons. Moreover, enhancement of autophagic flux was responsible for the protective effects of DNLA on axonal degeneration. DNLA prevents Aβ 25-35 -induced axonal degeneration via activation of autophagy process and could be a novel therapeutic target. © 2017 John Wiley & Sons Ltd.

  11. Induction of protein synthesis in Escherichia coli following UV- or γ-irradiation, mitomycin C treatment or tif expression

    International Nuclear Information System (INIS)

    West, S.C.; Emmerson, P.T.

    1977-01-01

    The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or γ-rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in the protein X. Synthesis of large quantities of protein X is induced by UV, γ-rays, MMC treatment or tif expression in rec + but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA + cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation. Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage lambda or septation. (orig./MG) [de

  12. Targeting tumor-initiating cells: Eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction

    Science.gov (United States)

    Lamb, Rebecca; Harrison, Hannah; Smith, Duncan L.; Townsend, Paul A.; Jackson, Thomas; Ozsvari, Bela; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

    2015-01-01

    We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than “bulk” cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to

  13. Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line

    International Nuclear Information System (INIS)

    O'Reilly, M.A.; Gazdar, A.F.; Clark, J.C.; Pilot-Matias, T.J.; Wert, S.E.; Hull, W.M.; Whitsett, J.A.

    1989-01-01

    Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression

  14. Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

    Science.gov (United States)

    Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

    2016-06-12

    Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

  15. Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii

    International Nuclear Information System (INIS)

    Silva, A.M. da; Costa Maia, J.C. da; Juliani, M.H.

    1986-01-01

    Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using ( 35 S)methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores. (Author)

  16. Is action potential threshold lowest in the axon?

    NARCIS (Netherlands)

    Kole, Maarten H. P.; Stuart, Greg J.

    2008-01-01

    Action potential threshold is thought to be lowest in the axon, but when measured using conventional techniques, we found that action potential voltage threshold of rat cortical pyramidal neurons was higher in the axon than at other neuronal locations. In contrast, both current threshold and voltage

  17. Local NSAID infusion does not affect protein synthesis and gene expression in human muscle after eccentric exercise

    DEFF Research Database (Denmark)

    Mikkelsen, U R; Schjerling, P; Helmark, Ida Carøe

    2010-01-01

    models, and inhibit the exercise-induced satellite cell proliferation and protein synthesis in humans. However, the cellular mechanisms eliciting these responses remain unknown. Eight healthy male volunteers performed 200 maximal eccentric contractions with each leg. To block prostaglandin synthesis...... locally in the skeletal muscle, indomethacin (NSAID) was infused for 7.5 h via microdialysis catheters into m. vastus lateralis of one leg. Protein synthesis was determined by the incorporation of 1,2-(13)C(2) leucine into muscle protein from 24 to 28 h post-exercise. Furthermore, mRNA expression...... of selected genes was measured in muscle biopsies (5 h and 8 days post-exercise) by real-time reverse transcriptase PCR. Myofibrillar and collagen protein synthesis were unaffected by the local NSAID infusion. Five hours post-exercise, the mRNA expression of cyclooxygenase-2 (COX2) was sixfold higher...

  18. Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

  19. Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3β signaling.

    Science.gov (United States)

    Shaw, Jillian L; Chang, Karen T

    2013-01-01

    Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD.

  20. Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

    Science.gov (United States)

    McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

    2014-01-24

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

  1. Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

    Science.gov (United States)

    McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

    2014-01-01

    Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

  2. Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

    Science.gov (United States)

    Tortosa, Elena; Hoogenraad, Casper C

    2018-02-01

    In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. 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.

  4. Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

    Directory of Open Access Journals (Sweden)

    Brita Singers Sørensen

    Full Text Available The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect, which weakens the spatial linkage between hypoxia and acidosis.Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15 were treated with hypoxia, acidosis (pH 6.3, or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe, genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2, and Ribosomal protein L37 (RPL37. Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa and protein synthesis (both cell lines was observed when hypoxia and low pHe were combined.We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de

  5. An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

    Czech Academy of Sciences Publication Activity Database

    Hronová, Vladislava; Valášek, Leoš

    2017-01-01

    Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 7.725, year: 2016

  6. Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

    International Nuclear Information System (INIS)

    Kopecky, J.; Baudysova, M.; Zanotti, F.; Janikova, D.; Pavelka, S.; Houstek, J.

    1990-01-01

    In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria

  7. Motor Axonal Regeneration After Partial and Complete Spinal Cord Transection

    Science.gov (United States)

    Lu, Paul; Blesch, Armin; Graham, Lori; Wang, Yaozhi; Samara, Ramsey; Banos, Karla; Haringer, Verena; Havton, Leif; Weishaupt, Nina; Bennett, David; Fouad, Karim; Tuszynski, Mark H.

    2012-01-01

    We subjected rats to either partial mid-cervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor (BDNF) gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared to several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair, and the need for additional control and shaping of axonal regeneration. PMID:22699902

  8. Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma.

    Science.gov (United States)

    Breen, Kevin T; Anderson, Sarah R; Steele, Michael R; Calkins, David J; Bosco, Alejandra; Vetter, Monica L

    2016-01-01

    Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs), and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1) mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b, and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2) expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1). There was also no significant increase in the proinflammatory

  9. MATHEMATICAL AND COMPUTATIONAL MODELLING OF RIBOSOMAL MOVEMENT AND PROTEIN SYNTHESIS: AN OVERVIEW

    Directory of Open Access Journals (Sweden)

    Tobias von der Haar

    2012-04-01

    Full Text Available Translation or protein synthesis consists of a complex system of chemical reactions, which ultimately result in decoding of the mRNA and the production of a protein. The complexity of this reaction system makes it difficult to quantitatively connect its input parameters (such as translation factor or ribosome concentrations, codon composition of the mRNA, or energy availability to output parameters (such as protein synthesis rates or ribosome densities on mRNAs. Mathematical and computational models of translation have now been used for nearly five decades to investigate translation, and to shed light on the relationship between the different reactions in the system. This review gives an overview over the principal approaches used in the modelling efforts, and summarises some of the major findings that were made.

  10. The induction of the oxidative burst in Elodea densa by sulfhydryl reagent does not depend on de novo protein synthesis

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    Amicucci, Enrica [Milan, Univ. (Italy). Dipt. di Fisiologia e Biochimica delle Piante

    1997-12-31

    In Elodea densa Planchon leaves, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a marked and temporary increase of respiration that is insensitive to cyanide, hydroxamate and propylgallate and completely inhibited by diphenylene iodonium (DPI) and by quinacrine. In this paper the author investigates whether the mechanism that causes the oxidative burst depends on the activation of preexisting oxidative systems or on the activation of de novo protein synthesis. The inhibitors used were cycloheximide (CHI) which inhibits protein synthesis in plant cells by depressing the incorporation of aminoacids into proteins and cordycepin, an effective inhibitor of mRNA synthesis. The data support the idea that the mechanism investigated depends on the activation of a long lived protein(s) and not on de novo protein synthesis.

  11. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    Science.gov (United States)

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  12. Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination.

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    Schultz, Verena; van der Meer, Franziska; Wrzos, Claudia; Scheidt, Uta; Bahn, Erik; Stadelmann, Christine; Brück, Wolfgang; Junker, Andreas

    2017-08-01

    Remyelination is in the center of new therapies for the treatment of multiple sclerosis to resolve and improve disease symptoms and protect axons from further damage. Although remyelination is considered beneficial in the long term, it is not known, whether this is also the case early in lesion formation. Additionally, the precise timing of acute axonal damage and remyelination has not been assessed so far. To shed light onto the interrelation between axons and the myelin sheath during de- and remyelination, we employed cuprizone- and focal lysolecithin-induced demyelination and performed time course experiments assessing the evolution of early and late stage remyelination and axonal damage. We observed damaged axons with signs of remyelination after cuprizone diet cessation and lysolecithin injection. Similar observations were made in early multiple sclerosis lesions. To assess the correlation of remyelination and axonal damage in multiple sclerosis lesions, we took advantage of a cohort of patients with early and late stage remyelinated lesions and assessed the number of APP- and SMI32- positive damaged axons and the density of SMI31-positive and silver impregnated preserved axons. Early de- and remyelinating lesions did not differ with respect to axonal density and axonal damage, but we observed a lower axonal density in late stage demyelinated multiple sclerosis lesions than in remyelinated multiple sclerosis lesions. Our findings suggest that remyelination may not only be protective over a long period of time, but may play an important role in the immediate axonal recuperation after a demyelinating insult. © 2017 The Authors GLIA Published by Wiley Periodicals, Inc.

  13. A developmental timing switch promotes axon outgrowth independent of known guidance receptors.

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    Katherine Olsson-Carter

    2010-08-01

    Full Text Available To form functional neuronal connections, axon outgrowth and guidance must be tightly regulated across space as well as time. While a number of genes and pathways have been shown to control spatial features of axon development, very little is known about the in vivo mechanisms that direct the timing of axon initiation and elongation. The Caenorhabditis elegans hermaphrodite specific motor neurons (HSNs extend a single axon ventrally and then anteriorly during the L4 larval stage. Here we show the lin-4 microRNA promotes HSN axon initiation after cell cycle withdrawal. Axons fail to form in lin-4 mutants, while they grow prematurely in lin-4-overexpressing animals. lin-4 is required to down-regulate two inhibitors of HSN differentiation--the transcriptional regulator LIN-14 and the "stemness" factor LIN-28--and it likely does so through a cell-autonomous mechanism. This developmental switch depends neither on the UNC-40/DCC and SAX-3/Robo receptors nor on the direction of axon growth, demonstrating that it acts independently of ventral guidance signals to control the timing of HSN axon elongation.

  14. Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

    International Nuclear Information System (INIS)

    Huang Jialing; Lazear, Helen M.; Friedman, Harvey M.

    2011-01-01

    The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

  15. The Molecular and Cellular Mechanisms of Axon Guidance in Mossy Fiber Sprouting

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    Ryuta Koyama

    2018-05-01

    Full Text Available The question of whether mossy fiber sprouting is epileptogenic has not been resolved; both sprouting-induced recurrent excitatory and inhibitory circuit hypotheses have been experimentally (but not fully supported. Therefore, whether mossy fiber sprouting is a potential therapeutic target for epilepsy remains under debate. Moreover, the axon guidance mechanisms of mossy fiber sprouting have attracted the interest of neuroscientists. Sprouting of mossy fibers exhibits several uncommon axonal growth features in the basically non-plastic adult brain. For example, robust branching of axonal collaterals arises from pre-existing primary mossy fiber axons. Understanding the branching mechanisms in adulthood may contribute to axonal regeneration therapies in neuroregenerative medicine in which robust axonal re-growth is essential. Additionally, because granule cells are produced throughout life in the neurogenic dentate gyrus, it is interesting to examine whether the mossy fibers of newly generated granule cells follow the pre-existing trajectories of sprouted mossy fibers in the epileptic brain. Understanding these axon guidance mechanisms may contribute to neuron transplantation therapies, for which the incorporation of transplanted neurons into pre-existing neural circuits is essential. Thus, clarifying the axon guidance mechanisms of mossy fiber sprouting could lead to an understanding of central nervous system (CNS network reorganization and plasticity. Here, we review the molecular and cellular mechanisms of axon guidance in mossy fiber sprouting by discussing mainly in vitro studies.

  16. Plexin A3 and turnout regulate motor axonal branch morphogenesis in zebrafish.

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    Rajiv Sainath

    Full Text Available During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch

  17. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    Science.gov (United States)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  18. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    Science.gov (United States)

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  20. A high mitochondrial transport rate characterizes CNS neurons with high axonal regeneration capacity.

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    Romain Cartoni

    Full Text Available Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. DRG neurons globally increase axonal transport during regeneration. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. Here, using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration.