Biomedical subjects

T B Shea

Publications and source records attributed to T B Shea.

At least 91 records · Page 5Linked to original sources

PubMed1992

Aluminum alters the electrophoretic properties of neurofilament proteins: role of phosphorylation state.

Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)-containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 microM) and was not reversed by 20% SDS, 6 M urea, freeze-thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high-molecular-weight neurofilament subunit (NF-H) entered SDS-containing gels after exposure to aluminum lactate but migrated aberrantly as a long high-molecular-weight streak. Migration of the 160-kDa alpha-chymotryptic cleavage product of NF-H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS-containing gels by AlCl3. Dephosphorylation of NF-H and the middle-molecular-weight neurofilament subunit (NF-M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF-H or NF-M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C-terminal sidearms of NFPs, resulting in intermolecular cross-linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication.

Aluminum↗

PubMed1992

Distinct mechanisms of differentiation of SH-SY5Y neuroblastoma cells by protein kinase C activators and inhibitors.

Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-beta-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-beta-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-beta-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-beta-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

Alkaloids↗

PubMed1991

Differential appearance of extensively phosphorylated forms of the high molecular weight neurofilament protein in regions of mouse brain during postnatal development.

The appearance and accumulation of extensively phosphorylated forms of the high molecular weight neurofilament protein (H-phos) was studied in six regions of mouse brain during postnatal development by quantitative immunoblot analyses. H-phos (migrating at 200 kDa) was detected in brainstem, cerebellum, cortex and hippocampus as early as postnatal day 1. While NF-H levels increased dramatically during subsequent postnatal development in these regions, and reached levels similar to those observed in adult brain by postnatal day 14, quantitative differences were observed in both the rate and the extent of increase among individual regions. The most rapid accumulation of H-phos was observed in brainstem and cortex, where H-phos increased within the first postnatal week to levels comparable to those of adult brain. However, H-phos exhibited a slower developmental change in cerebellum, where the levels increased uniformly over the first two postnatal weeks. In hippocampus, the major increase in H-phos levels was delayed until the second postnatal week. In contrast to its early detection in the above regions, H-phos was not detected in immunoblot analyses of olfactory bulb or hypothalamus cytoskeletons at postnatal day 1, indicating that in these regions the accumulated levels of posttranslationally modified forms of this protein appeared relatively late. Furthermore, H-phos levels in hippocampus did not level off at postnatal day 14 and continued to increase until at least postnatal day 21. Immunoblot analyses of whole embryonic brain revealed the presence of H-phos as early as embryonic day 17, demonstrating that some mouse brain regions carry out extensive phosphorylation of NF-H during embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Staurosporine-induced morphological differentiation of human neuroblastoma cells.

SH-SY-5Y human neuroblastoma cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of cAMP- and cGMP-dependent protein kinases but not protein kinase C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly, suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement of existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Transient neuritogenesis in NB2a/d1 neuroblastoma cells induced by glial-derived protease inhibitors.

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.

Animals↗

PubMed1991

Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells.

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.

Animals↗

PubMed1991

Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis.

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.

Animals↗

PubMed1991

A method for phospholipid-mediated delivery of specific antibodies into adherent cultured cells.

A simple and rapid procedure for the intracellular delivery of macromolecules into adherent cultured cells is described. Cells are incubated with cold glycerol, then transiently made permeable with L-alpha-lysophosphatidylcholine (LPC) in the presence of test compound to be loaded into cells. LPC induces temporary permeability of the plasma membrane, as evidenced by the loss and recovery of the cells' ability to exclude trypan blue. Molecules at least as large as antibodies are internalized during this transient permeability. Antibodies delivered intracellularly in this manner are able to complex with their specific antigen and exert functional consequences on normal cell metabolism, suggesting that this procedure is useful for determining protein function. As one example of this, we present data on the ability of specific antibodies, delivered intracellularly in this manner, to inhibit morphological differentiation (i.e., neurite outgrowth) in a neuroblastoma cell line.

Antibodies↗

PubMed1990

Transient increase in vimentin in axonal cytoskeletons during differentiation in NB2a/d1 cells.

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.

Animals↗

PubMed1990

Post-translational modification of alpha-tubulin by acetylation and detyrosination in NB2a/d1 neuroblastoma cells.

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.

Acetylation↗

PubMed1990

Alterations in dynamics of microtubule assembly during axonal neuritogenesis in NB2a/d1 cells.

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.

Animals↗

PubMed1990

Neuritogenesis in mouse NB2a/d1 neuroblastoma cells: triggering by calcium influx and involvement of actin and tubulin dynamics.

Ionophore (A23187)-mediated calcium influx induced rapid neurite outgrowth in NB2a/d1 cells. This outgrowth was prevented by colchicine but not by cycloheximide, demonstrating a requirement for microtubule assembly but not de novo synthesis. Cytochalasin B induced rapid, colchicine-sensitive outgrowth, indicating that depolymerization of the submembrane actin network may be sufficient to allow neurite outgrowth under conditions which permitted microtubule assembly. Neurites induced by serum-deprivation or calcium influx were rapidly retracted by colchicine unless cytochalasin B was first added, indicating that the actin network may provide the retractile force which mediates neurite retraction following microtubule depolymerization. We conclude that neurite outgrowth can be initiated in NB2a/d1 cells by calcium influx, and may involve alterations in actin and microtubule dynamics.

Actins↗

PubMed1990

Dynamics of phosphorylation and assembly of the high molecular weight neurofilament subunit in NB2a/d1 neuroblastoma.

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)

Antigens↗

PubMed1989

Appearance and localization of phosphorylated variants of the high molecular weight neurofilament protein in NB2a/d1 cytoskeletons during differentiation.

We used immunoblot and immunocytochemical methodologies to characterize the appearance and intracellular localization of the high molecular weight neurofilament subunit (NF-H) within the Triton-insoluble cytoskeleton during the first 5 days of differentiation of mouse NB2a/d1 neuroblastoma cells. Hypophosphorylated and partially phosphorylated forms of NF-H were detected in cells before and throughout differentiation. By contrast, some extensively phosphorylated forms of NF-H were first detected on the third day of differentiation and at least one additional 200 kDa isoform was visualized in cytoskeletons only after five days of differentiation. Extensively phosphorylated forms of NF-H were restricted to axonal neurites; by contrast, hypophosphorylated and partially phosphorylated forms of NF-H were present throughout undifferentiated and differentiated cells.

Animals↗

PubMed1989

Aluminum salts induce the accumulation of neurofilaments in perikarya of NB2a/dl neuroblastoma.

NB2a/dl neuroblastoma cells were exposed to aluminum chloride or aluminum lactate (0.1-1 mM) for 3 and 6 days. Additional cultures were exposed to aluminum salts as the cells were stimulated to elaborate axonal neurites by dibutyryl cyclic AMP. By phase-contrast microscopy, aluminum salts had no effect on the morphology of undifferentiated (NB2a(-] or differentiated (NB2a(+] cells, or on neuritic elaboration and maintenance. Silver straining by the Bielschowsky method, however, demonstrated argyrophilic accumulations in perikarya of many NB2a(-) and NB2a(+) cells treated with aluminum salts. At the ultrastructural level, whorls of intermediate filaments were the most prominent abnormalities in neuronal perikarya. Although phosphorylated high-molecular weight neurofilament subunits (NF-H) are normally detected by immunocytochemical analyses only within axonal neurites of NB2a/dl cells, aluminum salt treatment caused the detection of phosphorylated epitopes of NF-H within perikaryal of NB2a(-) and NB2a(+) cytoskeletons, suggesting that the argyrophilic filamentous accumulations are composed at least partly of phosphorylated NF-H.

Aluminum↗

PubMed1989

Expression of the plasma membrane proteolipid in mouse neuroblastoma cells: transient increase in synthesis during differentiation with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate.

We have examined the regulation of plasma membrane proteolipid (PM-PLP) synthesis and steady-state levels in mouse NB2a/d1 neuroblastoma cells during differentiation with dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA), agents which have been previously shown to induce the elaboration of exclusively axonal or dendritic neurites, respectively. We report that a PM-PLP-immunoreactive species is expressed by this neuroblastoma cell line, and that its expression is regulated by specific states of differentiation. Differentiation of cells with dbcAMP was accompanied by an initial 2-fold increase in this PM-PLP immunoreactive species at 24 h after treatment, which returned to control levels by 96 h after treatment. By contrast, no significant increase in synthesis was detected when cells were treated with RA. Protein blot analysis of PM-PLP in dbcAMP-treated cells indicated that there was little change in its steady-state level until 96 h following treatment, at which time a reduction of 40% was observed. Throughout induced differentiation with dbcAMP, NB2a/d1 cells continued to express a PM-PLP-immunoreactive species which comigrated on immunoblot analysis with PM-PLP form characteristic of embryonic brain (14-16 kDa), and apparently did not express the PM-PLP form characteristic of adult brain (18 kDa).

Animals↗

PubMed1988

Soluble, phosphorylated forms of the high molecular weight neurofilament protein in perikarya of cultured neuronal cells.

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.

Animals↗

PubMed1988

Neurofilament triplet proteins of NB2a/d1 neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation.

Induction of axonal neuritogenesis in NB2a/d1 cells was associated with an increased content of neurofilament proteins (NFPs) by immunoblot analysis. The major NFP subunits in differentiated [NB2a(+)] cells included microheterogenous forms with apparent molecular weights of 200-190 kDa (NFP-H), 143-142 kDa (NFP-M) and 70 kDa (NFP-L) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only NFP-L was detected in cytoskeletal preparations of undifferentiated [NB2a(-)] cells. All three NFPs of NB2a(+) cells incorporated 32P-orthophosphate in intact cells. A 160/155 kDa NFP-H immunoreactive polypeptide in NB2a(-) and NB2a(+) cells represented a relatively unmodified form of the 200 kDa NFP-H, since dephosphorylation of the 200 kDa NFP-H in vitro with alkaline phosphatase generated the 160/155 kDa forms. Triton-extracted NB2a(+) cells displayed NFP-H immunoreactivity in neurites and occasionally in perikaryal regions at the base of neurites. NFP-M was present throughout the neurites and somata of NB2a(+) cells, and was regularly detected in portions of perikarya in NB2a(-) cells. NFP-L immunoreactivity was distributed throughout the Triton-insoluble cytoskeleton of NB2a(-) and NB2a(+) cells. Immunocytochemical analyses revealed that extensively phosphorylated forms of NFP-H were largely restricted to the neurites of NB2a(+) cells, and less modified forms predominated throughout both perikarya and neurites of NB2a(-) and NB2a(+) cells.

Animals↗