Search PubMed⌕ Search

Biomedical subjects

N W Seeds

Publications and source records attributed to N W Seeds.

At least 37 records · Page 2Linked to original sources
PubMed1989

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.

Animals↗
PubMed1989

Characterization of 125I-tissue plasminogen activator binding to cerebellar granule neurons.

Mouse cerebellar cells in culture secrete tissue plasminogen activator (tPA) into the culture medium. Fibrin overlays have shown tPA to be associated with granule neurons in these cultures. This cell associated tPA can be displaced by extensive washing of the cells or by a brief lowering of the pH (less than 4), which leads to a loss of fibrinolytic activity by the cells. Incubation of these fibrinolytically inactive cells with exogenously added murine tPA leads to the restoration of the fibrinolytic activity, indicating the presence of tPA binding sites on these granule neurons. Using 125I-tPA, the binding to cerebellar granule neurons is rapid, saturable, specific, high affinity (Kd = 50 pM) and reversible. Both murine and human tPA compete with 125I-tPA for binding, with both murine and human urokinase (uPA) as well as human thrombin and plasminogen fail to compete. Neither the catalytic site nor the carbohydrate moiety of tPA appear to be involved in the binding, since both diisopropyl-fluorophosphate-treated tPA and endoglycosidase-H-treated tPA compete with 12I-tPA for binding. Furthermore, epidermal growth factor does not compete well with tPA for binding even at a 10:1 molar excess, suggesting that the epidermal growth factor-like (EGF) domain of tPA may not be involved in the binding mechanism. Autoradiography and antibody immunofluorescence show the specific tPA binding is to granule neurons in these cultures. Thus, granule neurons possess tPA receptors on their surface, where this protease binds retaining is functional activity and may play a role in cell and axon migration.

Animals↗
PubMed1988

Plasminogen activator secretion in relation to Schwann cell activities.

The molecular characterization of neural secretory plasminogen activators and their possible association with two well-defined Schwann cell activities (mitosis and migration) were investigated. Schwann cells from peripheral sensory ganglion roots were established as primary cultures of high purity. Conditioned culture medium was fractionated by gel electrophoresis and assayed for plasminogen-dependent in situ caseinolytic activity. Both tissue-type and urokinase-type plasminogen activators were detected early in culture. The amount of the tissue-type form decreased with increasing time in culture. Proliferative ability measured as [3H]thymidine incorporation into the nucleus varied under several culture conditions and was lowest in defined serum-free medium; however, these serum-free cultures expressed the highest level of plasminogen activator secreting cells. Combined autoradiography/fibrin overlay assays allowed for a direct analysis at the single cell level; interestingly the findings did not support the idea of an obligatory expression of the enzyme in replicating cells. Cell motility was assayed in combined gold particle clearing/fibrin overlay assays and likewise no obligatory association of protease secretion and cell movement could be demonstrated. The results are discussed in relation to previous studies in this field.

Animals↗
PubMed1986

Effect of proteases and their inhibitors on neurite outgrowth from neonatal mouse sensory ganglia in culture.

Developing neurons and Schwann cells have been shown to secrete proteases. The influence of these proteases on neurite outgrowth by cultured sensory ganglia was examined by adding specific protease inhibitors. Neonatal mouse dorsal root ganglia were cultured directly on tissue-culture plastic dishes in serum-free N2 medium with different protease inhibitors. Soybean trypsin inhibitor was found to double the extent of neurite outgrowth by 4 days in vitro. Ovomucoid trypsin inhibitor and leupeptin also increased neurite outgrowth, while alpha 1-antitrypsin, antipain and phenylmethylsulfonyl fluoride elicited a smaller effect. Furthermore, added trypsin or thrombin inhibited neurite outgrowth and the inhibition could be reversed by soybean trypsin inhibitor, while exogenous plasminogen or urokinase were inhibitory only at high concentrations. Thus neurite outgrowth probably requires a closely regulated system of protease secretion and protease inhibitor production.

Animals↗
PubMed1986

Transglutaminase and neuronal differentiation.

During mouse brain maturation cellular transglutaminase specific activity increases 2.5 fold from day 3 to adulthood. A more pronounced increase is seen during morphological differentiation of mouse neuroblastoma cells, where serum withdrawal induces neurite outgrowth concomitant with a 10 fold increase in transglutaminase specific activity. In contrast, non-dividing neuroblastoma cells lacking neurites show only a 1.5 fold increase in enzyme specific activity. Transglutaminase activity does not reach maximal levels until extensive neurite formation has occurred. More than 80% of the transglutaminase activity is found in the soluble component of brain and neuroblastoma homogenates. Using [3H]-putrescine as the acyl acceptor, endogenous acyl donor substrates in the neuroblastoma cells included proteins that comigrated on SDS-PAGE with tubulin and actin; however, very high molecular weight crosslinked material is the major reaction product in vitro. When purified brain tubulin, microtubule associated proteins and microtubules were compared as exogenous substrates, only the polymeric microtubules were a good acyl donor substrate. Furthermore, preincubation of purified tubulin with transglutaminase and putrescine stimulated both the rate and extent of microtubule assembly. These findings suggest that transglutaminase may mediate covalent crosslinking of microtubules to other cellular components, or the post-translational modification of tubulin by the formation of gamma-glutamylamines.

Aging↗
PubMed1986

Normal and malignant cells, including neurons, deposit plasminogen activator on the growth substrata.

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.

Animals↗
PubMed1986

Oligodendroglia development in cell culture as monitored with a monoclonal antibody.

A new marker for young oligodendrocytes has been identified by a monoclonal antibody (mOg-1, IgM isotype) prepared from cerebellar plasma membrane stimulated mouse lymphocytes. mOg-1 reactive cells in the mouse cerebellum first appear at day 19 of gestation. Future white matter layers of fixed sections of neonatal rat cerebellum were labeled with mOg-1. Although EM analysis has shown cell-surface binding by presumptive oligodendroglia in neonatal cerebellum, the antibody does not bind to compact myelin. In cell cultures prepared from 6-d-old mice, 1.1% of the cells bound mOg-1 after 3 d in culture, but up to 5% of the cells bound mOg-1 after 2 weeks in culture. Of these same Og-1-positive cells, 69% bound anti-galactocerebroside and 65% bound anti-myelin basic protein. After a week in culture Og-1-positive cells often produced lamellar sheets extending a millimeter over the polylysine substratum in the absence of normal myelin formation. mOg-1 recognizes a cell-surface determinant distinct from well-characterized oligodendroglial molecules (galactocerebroside, sulfatide and myelin basic protein) that is expressed early in oligodendrocyte development. The antibody has been used to follow the maturation of oligodendrocytes in cultures of both normal and jimpy mouse cerebellum.

Animals↗
PubMed1984

Peripheral neurons and Schwann cells secrete plasminogen activator.

The secretion of the protease plasminogen activator (PA) by cells of developing peripheral nerve was demonstrated. Fetal and early postnatal dorsal root ganglia were established in culture as explants or as individual neurons and Schwann cells. A fibrin overlay assay was used to visualize the locations of PA secretion. Fibrinolytic zones formed around the somata of explants and were skewed in the direction of maximal fiber outgrowth. Individual growth cones at the tips of long fasiculated fiber bundles also released PA. Approximately 50% of individual neurons showed PA secretion; especially pronounced release occurred at some growth cones. Culture of nerve growth factor-independent adult neurons showed that PA expression was independent of effects of this growth hormone. A subpopulation of Schwann cells was also active in PA secretion, which could be detected at the soma, at the bipolar processes, or along the entire cell length. Possible functions of neural PA in development and regeneration are discussed.

Animals↗
PubMed1983

Limited proteolysis of tubulin: nucleotide stabilizes an active conformation.

Limited proteolysis has been used to examine tubulin structure as related to microtubule assembly. Purified tubulin, freed of exchangeable nucleotide, was digested with low concentrations of chymotrypsin (0.01-0.1% w/w to tubulin) and its polymerization behavior investigated. Chymotryptic proteolysis resulted in a loss of assembly activity with apparent first-order inactivation kinetics. The inactivation rates were dependent on both chymotrypsin concentration and incubation temperature. However, these conditions of proteolysis did not significantly affect tubulin's colchicine binding activity. Polyacrylamide-NaDodSO4 electrophoresis demonstrated the major cleavage fragments of tubulin to be 34 and 17 kilodaltons. Furthermore, amino-terminal analysis showed methionine for the 17-kilodalton fragment and both glutamate and serine for the 34-kilodalton fragment. Microtubular structures formed from chymotryptic tubulin possessed constrictions and had a frayed appearance in the electron microscope; these polymers were composed of both native tubulin and the 34- and 17-kilodalton fragments, suggesting that the loss of microtubule assembly results from tubulin cleavage and the altered interaction of cleavage fragments with uncleaved tubulin subunits. Interestingly, the readdition of GTP prior to proteolysis significantly protected tubulin's assembly capacity, presumably by stabilizing the fragments in an active conformation as indicated by circular dichroism spectra.

Animals↗
PubMed1983

Affinity labeling of tubulin's exchangeable guanosine 5'-triphosphate binding site.

Tubulin requires GTP for maximal rate and extent of polymerization into microtubules. The localization of the guanine nucleotide in the microtubule was examined by preparing affinity probes that would permit tubulin polymerization prior to their covalent coupling to amino acids in tubulin's exchangeable GTP binding site. Two different hydrolyzable GTP analogues with modified ribose moieties, 3'-p-azidobenzoyl)-GTP and the periodate oxidation product of GTP, 2-(guanylformylmethoxy)-3-(triphospho)propanal, were isolated by thin-layer chromatography and high-voltage electrophoresis and identified by ultraviolet and infrared spectroscopy. The analogues bind to the tubulin molecule and promote polymerization. After tubulin polymerization and isolation of microtubules, the [3H]GTP analogues were covalently coupled to tubulin by NaBH4 reduction or UV irradiation. The microtubules possessed about 1 mol of acid-precipitable 3H-labeled nucleotide/mol of tubulin dimer. Separation of the subunits showed that the nucleotide analogues were associated with both alpha and beta subunits of tubulin in nearly equal amounts. The binding of analogues to both alpha and beta subunits was saturable and competitive with GTP. Cyanogen bromide cleavage of both alpha and beta subunits showed that the 3H-labeled nucleotide was associated with a single molecular weight species of similar size (approximately 10 000) from each subunit. Two-dimensional electrophoresis of chymotryptic peptides from both (alpha and beta) cyanogen bromide fragments showed that the 3H-labeled nucleotide was associated with a peptide of nearly identical migration properties from both subunits. These results suggest that a similar peptide segment of both alpha- and beta-tubulin has the ability to bind GTP. Furthermore, this peptide was localized to the amino-terminal one-third of the tubulin molecule.

Affinity Labels↗
PubMed1983

Cerebellar plasma membrane proteins and their antisera.

Plasma membranes have been isolated from neonatal through adult cerebella by a sequence of differential centrifugation, aqueous two-phase polymer fractionation and density gradient centrifugation. The protein composition of cerebellar membranes from various aged mice was compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Increases in the relative amount of membrane proteins with molecular weights (X 10(-3)) of 400, 340, 270, 220, 54, 44, and 9.5 were most pronounced, while a protein of 66,000 Mr disappeared between birth and Day 25. The relationship of these proteins and others to specific cell types in the cerebellum was examined by preparing membrane fractions from isolated granule and Purkinje cells, as well as from the cerebella of neurological mutant mice: reeler, weaver, staggerer, and nervous. In addition, those membrane proteins on the surface of dissociated cerebellar cells were identified by lactoperoxidase-catalyzed iodination, while glycoproteins were identified by galactose oxidase treatment and NaB3H4 reduction. Rabbit antisera were prepared toward those SDS-PAGE membrane proteins which appeared cell specific or developmentally regulated. Sera from these rabbits were used with indirect immunoperoxidase and immunofluorescence to stain frozen sections of mouse cerebellum and dissociated cerebellar cell cultures. In tissue sections antiserum toward the 400,000 Mr protein (p400) and antiserum p14.7 gave strong reactions with Purkinje cells while anti-p130 reacted preferentially with Purkinje cell somas, anti-p220 stained small cells in the internal granule layer and anti-p30 displayed a coarse, grainy staining of the granule and molecular layers, characteristic of a synaptic localization. Only anti-p220 and anti-p130 bound to freshly dissociated cells or cultured cerebellar cells. Large phase-bright cells in the cultures bound antiserum p130. Anti-p220 reacted specifically with a subpopulation of small round viable cells that bound tetanus toxin and decreased in number from 9% at Day 3 to 0.5% of the cells by Day 11, suggestive of granule neurons.

Age Factors↗
PubMed1982

Residual nucleotide and tubulin's ability to polymerize with nucleotide analogs.

The nucleotide requirements for microtubule assembly following two different methods of nucleotide removal were defined. Microtubular protein extracted with charcoal in a buffer containing 1 mM EDTA showed an absolute dependence on GTP for assembly, whereas, microtubular protein extracted with charcoal in a buffer containing 1 mM MgCl2 could assemble with the nucleotide analogs, guanosine-5'-(beta,gamma-imido)triphosphate (GMP-PNP) and guanosine-5'-(beta-gamma-methylene)triphosphate. Assembly promoted by the nucleotide analogs proceeded at slower rate and to a lesser extent than the assembly induced by GTP. Furthermore, after the analog-induced assembly had reached a steady state level, the addition of GTP immediately induced further tubulin assembly to the level produced by GTP alone. Microtubules assembled with GMP-PNP or GTP contained approximately 2.8 mol of bound nucleotide per mol of dimer. The two different methods of charcoal extraction produced microtubular protein preparations that differed in their residual nucleotide and Mg2+ content. The EDTA-charcoal-extracted microtubular protein retained 1 mol of tightly bound nucleotide (GDP) and 1 mol of Mg2+ per mol of dimer, whereas, the Mg2+ charcoal-extracted microtubular protein retained 1.4 mol of bound nucleotide (1.0 mol of GDP + 0.4 mol of GTP) and 1.5 mol of Mg2+ per mol of dimer. The results suggest that only that fraction of tubulin subunits containing bound GTP can assemble with the nucleotide analogs and that microtubules may possess a third or polymer binding site for nucleotide.

Animals↗
PubMed1982

Development of ion metabolism in reaggregated brain cell cultures.

Mouse brain cell reaggregates have been used to study changes in sodium- and potassium-dependent ouabain-sensitive adenosine phosphohydrolase (Na+, K+-ATPase) activity and in 86Rb+ uptake and exit during development. Na+, K+-ATPase activity in these cultures has two ouabain-inhibitable components, both of which increased severalfold between day 3 and day 17 in culture. This increase, however, was less than that in developing brain. Little change in either total or extracellular water or in the equilibrium levels of Na+ and K+ occurred during development. The uptake of 86Rb+ measured a 10-min incubation showed only a modest increase during culture, whereas the exit of 86Rb+ from reaggregates preloaded with the tracer increased approximately fourfold. The exit consisted of both K+-independent and K+-stimulated components and the K+-stimulated fraction contributed most of the developmental change. When uptake rates were corrected for the contribution of the developmental changes in exit, these rates were found to increase as well. The 86Rb+ uptake correlated closely with the activity of the Na+,K+-ATPase during development. The pattern of developmental changes in enzyme activity and 86Rb+ uptake and exit suggest that, while little change in the steady-state levels of the ions occurred, the rates of ion movement increase markedly.

Animals↗
PubMed1981

Plasminogen activator release at the neuronal growth cone.

The site of plasminogen activator release by differentiated neuroblastoma clonal cell lines was determined with a fibrin overlay assay. Release of plasminogen activator was seen at the growth cone in 72 percent of the cells bearing neurites. For 21 percent of these cells the growth cone was the predominant or exclusive site of this enzyme activity. Selective release of protease at the "trailblazing" tip of the neurite may be important in neuron migration and neurite growth in vivo.

Animals↗
PubMed1981

Plasminogen activator secretion by granule neurons in cultures of developing cerebellum.

Dispersed cell cultures were established from 7- to 9-day postnatal mouse cerebellum. The fibrin slice method was used to obtain a localization of plasminogen activator production to specific cells. Fibrinolytically active cells were small (5- to 8-micrometer diameter), round, and occurred singly or in aggregates. Fibrinolysis was both plasminogen and time dependent, inhibitable by epsilon-aminocaproic acid and soybean trypsin inhibitor and did not occur when cells were fixed in formalin prior to the fibrin overlay. Strong fibrin degradation occurred only when granule neurons were abundant in the cultures. These plasminogen activator secreting cells were identified as granule neurons by cell separation methods, nuclear morphology, and their ability to bind tetanus toxin and rabbit antiserum against mouse cerebellum (anti-Cbl-1 antiserum). Plasminogen activator also could be quantified in fractionated tissue homogenates or in cell culture medium by the 125I-labeled fibrin plate assay. Fibrinolysis in cerebellar extracts was 95% dependent on the presence of added plasminogen; furthermore, the activity was greater in cerebellar extracts as compared to cerebral cortex of the same age. At the age examined, the cerebellum contains many migratory neurons, and plasminogen activator production may be involved in the process of cell movement.

Animals↗
PubMed1979

Sulfatide synthesis by neural cell lines.

Several neural cell lines were examined for their ability to synthesize sulfatide and 2',3'-cyclic nucleotide phosphohydrolase, biochemical components characteristic of myelin. The mouse glioma G26 and the rat schwannoma TRM6B actively produced sulfatide, while the rat glioma C6 was inactive, supporting the probable oligodendroglial origin of the G26. In contrast, the C6 cell line had a high level of 2'-3'-cyclic nucleotide phosphohydrolase activity, while TRM6B showed 30% and the G26 75% lower activities. Thus, these two activities appear to be independently regulated.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗