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John T. Roehrig - One of the best experts on this subject based on the ideXlab platform.
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Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice
Journal of neurovirology, 2009Co-Authors: Elizabeth Hunsperger, John T. RoehrigAbstract:West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with Nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log10 greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in Nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by...
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Characterization of West Nile viral replication and maturation in peripheral neurons in culture
Journal of NeuroVirology, 2005Co-Authors: Elizabeth Hunsperger, John T. RoehrigAbstract:The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of ≥10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans -Golgi inhibitor) or nocadazole (a β-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but Nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.
Elizabeth Hunsperger - One of the best experts on this subject based on the ideXlab platform.
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Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice
Journal of neurovirology, 2009Co-Authors: Elizabeth Hunsperger, John T. RoehrigAbstract:West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with Nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log10 greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in Nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by...
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Characterization of West Nile viral replication and maturation in peripheral neurons in culture
Journal of NeuroVirology, 2005Co-Authors: Elizabeth Hunsperger, John T. RoehrigAbstract:The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of ≥10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans -Golgi inhibitor) or nocadazole (a β-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but Nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.
Marjatta Raudaskoski - One of the best experts on this subject based on the ideXlab platform.
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Effects of Nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomycete Schizophyllum commune
Protoplasma, 1995Co-Authors: Ivan Rupes, Wei-zhi Mao, Helena Åström, Marjatta RaudaskoskiAbstract:The effects of Nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h Nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some Nocodazole-treated hyphae. After 2 h growth in the presence of Nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with Nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.
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Microtubule cytoskeleton in hyphal growth. Response to Nocodazole in a sensitive and a tolerant strain of the homobasidiomycete Schizophyllum commune.
European journal of cell biology, 1994Co-Authors: Marjatta Raudaskoski, Wei-zhi Mao, Tapani Yli-mattilaAbstract:In the wild-type strains of the homobasidiomycete Schizophyllum commune microtubules were totally depolymerized by low concentrations of Nocodazole, while high concentrations of benomyl only modified the structure of microtubule cytoskeleton. In the Nocodazole-tolerant mutant strain NT30 the microtubule cytoskeleton remained partly functional at a Nocodazole concentration which demolished the microtubules in the wild-type strains. The continuation of apical growth for several hours in the wild-type strain without cytoplasmic microtubules indicated that microtubules are not the major elements in hyphal extension growth. However, the irregular branching of the treated apical cells both in the Nocodazole-sensitive and -tolerant strain suggested that an intact microtubule cytoskeleton is needed for maintaining the direct extension of the leading hyphae at the colony edge. In the Nocodazole-sensitive strain growth in the absence of polymerized microtubules frequently led to the death of the apical cells even when the drug was removed. In the tolerant strain the nuclear divisions continued in spite of Nocodazole, but the uninucleate hyphal compartments became multinucleate. This probably resulted from poor segregation of nuclei and septation of hyphae at telophase, which indicated that these processes might be dependent on proper polymerization of cytoplasmic microtubules in higher fungi. The different electrophoretic mobility of the beta-tubulin from the NT30 strain and its parental strains suggested that the tolerance of the NT30 to Nocodazole could be due to a mutation in a beta-tubulin encoding gene.
J. T. Roehrig - One of the best experts on this subject based on the ideXlab platform.
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Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice
Journal of NeuroVirology, 2009Co-Authors: E. A. Hunsperger, J. T. RoehrigAbstract:West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with Nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log_10 greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in Nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by real-time reverse transcriptasepolymerase chain reaction (RT-PCR), did not occur until day 7. At these later time points (7 and 9 days PI), Nocodazole-treated WNV-infected animals attained viral titers in these tissues similar to titers in the untreated WNV-infected control animals. These results demonstrate that a single dose of Nocodazole delays, but does not block, WNV infection of the brain.
Lynne Cassimeris - One of the best experts on this subject based on the ideXlab platform.
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Nanomolar concentrations of Nocodazole alter microtubule dynamic instability in vivo and in vitro.
Molecular biology of the cell, 1997Co-Authors: R. J. Vasquez, Bonnie Howell, A.-m. C. Yvon, P. Wadsworth, Lynne CassimerisAbstract:Previous studies demonstrated that nanomolar concentrations of Nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a Nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of Nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of Nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in Nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that Nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM Nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with Nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after Nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that Nocodazole increases tubulin GTPase activity, we propose that Nocodazole acts by generating TuD subunits that then alter dynamic instability.
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Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes.
Journal of Cell Science, 1991Co-Authors: Lynne Cassimeris, Edward D. SalmonAbstract:The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and Nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM Nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/− 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in Nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–90 s in Nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM Nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.