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Biomedical subjects

N W Seeds

Publications and source records attributed to N W Seeds.

At least 55 records · Page 3Linked to original sources
PubMed1978

Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.

Animals↗
PubMed1978

Cell junction and ultrastructural development of reaggregated mouse brain cultures.

The morphological development of reaggregated embryonic mouse brain cells was followed by scanning and transmission electron microscopy. Initially, the reaggregated cells are loosely packed and lack specialized cell junction; however, after 2-4 days puncta adhaerens junctions are numerous. The first immature synapses are seen at 8-9 days of culture. These synaptic complexes mature in appearance and increase approximately 3-fold in number during the next 3 weeks of culture. Two distinct, electron dense, intercellular specialization, apparently unique to neural cell cultures, are observed in these older cultures. Brain cell reaggregates observed at 2, 3, and 5 months of culture show signs of aging, i.e., a gradual decrease in the number of synapses, myelin degeneration and increased lipofuscin granules.

Aging↗
PubMed1977

Stoichiometry of GTP hydrolysis and tubulin polymerization.

Microtubule formation from lamb brain tubulin isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethylenediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of microtubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 10(4) M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization. Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found.

Animals↗
PubMed1975

Expression of differentiated activities in reaggregated brain cell cultures.

Dissociated fetal mouse brain cells are allowed to reassociate in rotation culture to form aggregates. After several weeks these reaggregated brain cell cultures show markedly increased specific activities of monoamine oxidase, lactate dehydrogenase, and the brain-specific protein S-100, while catechol-O-methyltransferase activity increases slightly. Similar changes in these activities are found during mouse brain maturation. The amounts of monoamine oxidase, catechol-O-methyltransferase, and S-100 were also determined in surface cultures of fetal mouse brain cells, as well as glioma and neuroblastoma cell lines. The fetal brain and glial cell cultures possess much higher activities than the cultured neuroblastoma cells. However, lactate dehydrogenase activity was highest in the glioma and lowest in the surface cultures of fetal brain cells.

Age Factors↗
PubMed1975

Cerebellar cell surface antigens of mouse brain.

Reaggregated cells from 6- to 8-day-old mouse cerebella have been used to raise antibodies in rabbits. The interaction of these antibodies with cerebellar cell surface components was assessed by cytotoxicity of 51Cr-labeled cerebellar cell cultures and indirect immunofluorescence. A quantitative comparison of the relative amount of antigen on cells from other mouse tissues, brain regions, cerebella of various aged mice and mutant mice, and other animal species, as well as several clonal cell lines of nervous system origin, was made. A fixed subthreshold concentration of antiserum was adsorbed with increasing numbers of dissociated cells or amounts of particulate tissue prior to incubation with complement and 51Cr-labeled cerebellar target cells. Mouse thymus, spleen, liver, and heart tissue possess negligible adsorbing capacity, whereas kidney and sperm gave some adsorption. Of the brain regions examined, only cerebellum removed all immunofluorescence and cytotoxic activity, whereas other regions removed less than 90%, suggesting the possibility of cerebellar specific antigens on certain cell types. Only mouse and rat cerebellum gave measurable adsorptions, and this capacity decreased with increasing age. Although cerebellar mutants (stagger, weaver, and nervous) possessed similar adsorptive capacity, glioma and neuroblastoma clonal cell lines differed measurably in their adsorption; only the mouse neuroblastoma clones displayed significant adsorption of the antiserum.

Animals↗
PubMed1975

Tubulin constancy during morphological differentiation of mouse neuroblastoma cells.

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.

Cell Differentiation↗
PubMed1971

Norepinephrine stinulated increase of cyclic AMP levels in developing mouse brain cell cultures.

Norepinephrine causes a four- to sixfold increase in the intracellular level of cyclic AMP (adenosine 3', 5'-monophosphate) reaggregated brain cell cultures derived from embryonic mouse brain. The cyclic AMP level of adult brain is increased by norepinephrine; however, embryonic mouse brain does not show a cyclic AMP response. The aggregate cultures thus demonstrate an event of differentiation very similar to that seen in vivo.

Age Factors↗
PubMed1971

Synaptogenesis in reaggregating brain cell culture.

Dissociated cells from embryonic mouse brain reassociate in rotation culture to form highly organized aggregates. Electron microscopic observations of aggregates show the presence of synapses that mature and increase in number during culture. Apparent myelination of axons is observed in the aggregates after 5 weeks of culture. Thus, brain cell aggregates perform two highly specialized morphological events that are characteristic of mouse brain differentiation.

Animals↗
PubMed1971

Biochemical differentiation in reaggregating brain cell culture.

Dissociated cells from embryonic mouse brain reassociate in rotation culture to form aggregates. During cell culture the specific activities of choline acetyl-transferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and glutamate decarboxylase (EC 4.1.1.15) in the aggregates increase up to twenty-fold, a phenomenon that approximates some of the biochemical events in the development of the mouse brain.

Acetylcholinesterase↗
PubMed1970

Regulation of axon formation by clonal lines of a neural tumor.

Clonal lines of neuroblastoma cells were found to extend or retract axons depending upon the concentration of serum. Neurite extension was not inhibited by cycloheximide but was sensitive to colchicine or vinblastine, suggesting that neurite formation is dependent upon the assembly of microtubules or neurofilaments from preformed protein subunits.

Axons↗