Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The emulsifier for cyclosporin in clinical i.v. formulations, Cremophor EL, has recently come into question as a possible source of neurotoxic side effects in immunosuppressant therapy. To address this issue we tested Cremophor EL and cyclosporin on an in vitro neuronal model, the differentiating N1E.115 neuroblastoma cell. In terms of effects on elaboration of neurites by these cells, Cremophor accounted for nearly all the neurotoxicity of clinically formulated cyclosporin. At a concentration of 0.005% (v/v), Cremophor EL halved the number of cells that extended neurites after 48 hr in serum-free medium. Average neurite length was also reduced substantially. Inhibition of neurite outgrowth first became apparent 24 hr after exposure to Cremophor EL. Neurites that did grow in the presence of Cremophor were disfigured by a series of regularly spaced, gross dilatations (beads) filled with large (0.2-0.5 microns) lipid vesicles. Abnormalities of rapid axonal transport were documented in the beaded neurites by means of video-enhanced contrast, differential interference-contrast microscopy. Velocity of retrograde transport remained normal, but the velocity of anterograde transport and the total bidirectional flux of organelles were both reduced. It seems likely that the inhibition of neurite outgrowth, the swellings of the neurites and the abnormalities of transport are interrelated phenomena.
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PMID:Emulsifier for intravenous cyclosporin inhibits neurite outgrowth, causes deficits in rapid axonal transport and leads to structural abnormalities in differentiating N1E.115 neuroblastoma. 157 86

The neuronal growth-associated protein GAP-43 is expressed during axonal outgrowth and regeneration (for review, see Benowitz and Routtenberg, 1987). In the present study, we demonstrate that GAP-43 is constitutively expressed by NB2a/d1 neuroblastoma cells. The initial, most rapid outgrowth period of neuritogenesis [0-4 hr after dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) treatment] is accompanied by intense GAP-43 immunoreactivity along the entire length of most neurites. However, this immunoreactivity declined nearly to background levels within hours during continued neurite outgrowth and persisted only at varicosities and growth cones. GAP-43 was detectable by metabolic labeling and immunoblot analysis in undifferentiated cells, and synthetic rates and steady-state levels of GAP-43 underwent only a modest (approximately twofold) increase during dbcAMP-induced differentiation. Unlike levels observed in neurites, perikarya of undifferentiated and differentiated cells contained similar, intense levels of GAP-43 immunoreactivity. Neurite elaboration and GAP-43 immunoreactivity were unaffected by treatment with cycloheximide, suggesting that translocation of perikaryal GAP-43 pools, rather than de novo synthesis, contributes to the transient burst of GAP-43 observed in developing neurites. Phosphatidylcholine-mediated delivery of anti-GAP-43 antibodies (alpha GAP) into cells immediately before dbcAMP treatment arrested neuritogenesis but did not induce the retraction of existing neurites. These results indicate that, while GAP-43 expression is insufficient to induce neuritogenesis in NB2a/d1 cells, GAP-43 is nevertheless essential for the initial, dynamic phase of neurite outgrowth.
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PMID:Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis. 164 99

The initial outgrowth of neuritogenesis in mouse NB2a/d1 neuroblastoma cells may be regulated by thrombin or a thrombin-like protease, present either in serum or adsorbed to the plasma membrane, since neuritogenesis is induced by serum deprivation and treatment with the specific thrombin inhibitor, hirudin (Shea et al., 1991, J. Neurochem., 56:842). Cultured astroglial cells secrete factors that promote neuritogenesis, including protease inhibitors active against thrombin, leading to suggestions that the inhibition of specific neuronal surface proteases by the surrounding glial environment may represent an initial step in axonal outgrowth in situ. To examine the relative importance of glial-derived protease inhibitory activities on neurine outgrowth, we tested the neurite promoting effect of glial-conditioned medium (GCM) on NB2a/d1 cells. Like serum deprivation and hirudin treatment, GCM induced neurite outgrowth within 4 hr. Exogenous thrombin inhibited the effect of GCM, and cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM, suggesting that GCM induces neuritogenesis by inhibition of a thrombin-like protease. Unlike neurites induced by serum removal or hirudin addition, which are rapidly resorbed following serum replenishment or hirudin depletion, however, GCM-induced neurites continued to elongate after GCM removal. Furthermore, cultures treated simultaneously with GCM and thrombin exhibited delayed outgrowth of neurites following GCM removal which were insensitive to further thrombin treatment. These findings indicate that the initial elaboration of neurites can be mediated by glial-derived protease inhibitor(s) active against a thrombin-like protease, but indicate the requirement of additional glial-derived factors for the maintenance and continued elaboration of these neurites.
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PMID:Transient neuritogenesis in NB2a/d1 neuroblastoma cells induced by glial-derived protease inhibitors. 191 50

During dibutyryl cyclic AMP (dbcAMP)-mediated differentiation, axonal neurites elaborated by mouse NB2a/d1 neuroblastoma cells are initially colchicine-labile but attain colchicine-stability after 7 days. To examine whether or not differences in tubulin subunit turnover could account for the development of colchicine-stability, anti-tubulin antibodies were delivered into NB2a/d1 cells at various times during dbcAMP-mediated neurite outgrowth. These antibodies prevented initial neurite elaboration, and induced neurite retraction in cells treated with dbcAMP for up to 3 days, but did not induce neurite retraction for cells treated for 7 days. We conclude that a less dynamic, more slowly-turning over population of microtubules develops within neurites of cells treated with dbcAMP for 7 days.
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PMID:Alterations in dynamics of microtubule assembly during axonal neuritogenesis in NB2a/d1 cells. 196 29

Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.
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PMID:Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells. 199 97

The localization of vimentin (Vm) within the Triton-insoluble cytoskeleton was characterized during differentiation of mouse NB2a/dl neuroblastoma cells. Vm staining increased within neurites during the first day of differentiation, and then rapidly declined in both perikarya and neurites. By contrast, immunoreactivity against extensively phosphorylated forms of the high molecular weight neurofilament subunit (NF-H) was absent until the third day after differentiation. Immunoblot analyses confirmed that these alterations reflected specific changes in Vm and NF-H steady-state levels. Metabolic labeling demonstrated a decrease in the rate of Vm synthesis by the third day of differentiation. We conclude that changes in incorporation of intermediate filament species into the axonal cytoskeleton reflect distinct stages in neurite outgrowth and maturation; i.e., the Vm filament system may participate in initial stages of neuritogenesis during which outgrowth is most rapid, while NFPs may subsequently function in the establishment of a stabilized axonal cytoskeleton.
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PMID:Transient increase in vimentin in axonal cytoskeletons during differentiation in NB2a/d1 cells. 220 72

In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/d1 neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dynamics of phosphorylation and assembly of the high molecular weight neurofilament subunit in NB2a/d1 neuroblastoma. 221 24

Western blot analyses of total assembled microtubule fractions from NB2a/d1 neuroblastoma cells demonstrated that these cells are capable of post-translationally modifying alpha-tubulin by acetylation and detyrosination. Immunocytochemical analyses of NB2a/d1 cells differentiated with dbcAMP which had been processed under microtubule-stabilizing conditions demonstrated that all forms of alpha-tubulin were present throughout perikarya and neurites. By contrast, extraction of cells with Triton X-100 revealed a regional concentration of acetylated and detyrosinated alpha-tubulin subunits within axonal neurites, detectable in some cells after 3 days of differentiation and in nearly all cells after 7 days. Resistance of neurites to retraction following colchicine-treatment developed at a similar rate; furthermore, colchicine-resistant neurites contained intense acetylated alpha-tubulin immunoreactivity. We conclude that NB2a/d1 cells are capable of acetylating and detyrosinating alpha-tubulin subunits and that selective post-translational modification of alpha-tubulin subunits may be related to neuritic maturation.
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PMID:Post-translational modification of alpha-tubulin by acetylation and detyrosination in NB2a/d1 neuroblastoma cells. 232 28

Neutral endopeptidase-24.11 (EC 3.4.24.11) (NEP) is a transmembrane metallo-endopeptidase that has been shown to be involved in the degradation of several mammalian neuropeptides, including enkephalins. The enzyme has recently been found to be specifically associated with the axonal and synaptic membranes of neurons in the globus pallidus of the pig brain. This result suggests that neurons must possess mechanisms for targeting NEP to particular membrane domains. Study of these mechanisms would greatly benefit from the existence of an established neuron-like cell line capable of expressing and targeting NEP to specific membrane domains. For this reason we have used a retroviral vector containing the cDNA for rabbit kidney NEP to express this enzyme in a mouse neuroblastoma cell line (Neuro2A). Labelling of the cell surface with an antibody coupled to colloidal gold particles and examination of the cells by electron microscopy revealed a non-uniform distribution of NEP at the surface of the cells, the protein being preferentially associated with the membrane of neurites compared with the cell body. This observation suggests that Neuro2A cells possess a mechanism for targeting NEP to specific domains of the plasma membrane. This cell line could thus constitute a good model for studying the mechanisms responsible for targeting this enzyme to specialized regions of the plasma membrane.
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PMID:Recombinant neutral endopeptidase-24.11 expressed in mouse neuroblastoma cells is associated with neurite membranes. 233 3

The high molecular weight subunit of neurofilaments (NF-H) in mouse NB2a/d1 neuroblastoma cells is extensively phosphorylated and exhibits an apparent molecular weight of 200 kDa by SDS gel electrophoresis. In this study, we observed that extensively phosphorylated NF-H variants exist as both Triton-soluble and -insoluble forms, which display different cellular distributions. Perikarya and neurites of differentiated NB2a/d1 cells were immunostained by a polyclonal antiserum (anti-NF-H) that specifically recognizes the extensively phosphorylated NF-H forms and a monoclonal antibody (SMI-31) that recognizes phosphorylated epitopes of neurofilament proteins (NFPs). When cells were extracted with Triton X-100 to remove soluble proteins, however, only axonal neurites remained immunoreactive. Immunoblot analyses established the specificity of anti-NF-H and SMI-31 and demonstrated that both Triton-soluble and -insoluble NF-H subunits exhibit an apparent molecular weight of 200 kDa. Incorporation of radiolabeled phosphate into Triton-soluble NF-H following incubation of intact NB2a/d1 cells with 32P-orthophosphate confirmed that the Triton-soluble form of NF-H is a phosphoprotein. Most NF-H subunits in the Triton-soluble fraction sedimented after centrifugation at 100,000 g for 1 h, indicating that they may be present as oligomers. The implications of these data for the development of neurofibrillary pathology are discussed.
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PMID:Soluble, phosphorylated forms of the high molecular weight neurofilament protein in perikarya of cultured neuronal cells. 246 97


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