The growth-promoting and/or differentiation-blocking activities of Kirsten (Ki-MSV) or Harvey murine sarcoma virus (Ha-MSV) on various types of cells in vitro are well documented. Here we report an unexpected effect of these viruses on a rat phaeochromocytoma cell line, PC12. PC12 cells, which multiply indefinitely in growth medium, are known to respond to nerve growth factor (NGF) by cessation of cell division and expression of several properties resembling those of differentiated sympathetic neurones. We have found that Ki- and Ha-MSV mimic some, if not all, of the activities of NGF in PC12 cells, and there is evidence that the viral oncogenes, v-Ki-ras and v-Ha-ras, are responsible for this phenomenon. This system may be of value for studying the mechanism of action of the v-ras genes as well as the regulatory mechanism of growth and differentiation in neuronal cells.
Neuroblastoma clones were examined for choline acetyltransferase (EC 2.3.1.6), tyrosine hydroxylase (EC 1.14.3.a), acetylcholinesterase (EC 3 The neuroblastoma system established by Augusti-Tocco and Sato (1) provides an unusual opportunity to explore steps in neuron differentiation and function. The cells multiply rapidly in vitro, yet exhibit many properties characteristic of differentiated neurons (2-7). In this report, the properties of additional clones derived from the mouse neuroblastoma are described. Three cell types, cholinergic cells, adrenergic cells, and cells that do not synthesize acetylcholine or catecholamines, were detected. METHODS AND MATERIALSCells. Mouse neuroblastoma C-1300 cells were grown as described (7). Some clones were obtained in two stages: first, a well-isolated colony of cells in agar was picked and then cloned by isolation of a single cell with a stainless-steel cylinder. In other cases, cells were added to petri dishes containing broken coverslips; each glass shard with a single cell was then transferred to a separate dish.Chromosomes were analyzed by incubation of cells in logarithmic growth for 6-12 hr with 15-300 1M colcemide (N-desacetyl-N-methyl-colchicine) obtained from Ciba;chromosomes were spread by the method of Merchant, Kahn, and Murphy (8).Choline Acetyltransferase (EC 2.3.1.6) Assay. Cell monolayers were washed 3 times with an isotonic salt solution; then cells and protein were harvested by scraping and washing with 10 mM potassium phosphate buffer (pH 6.8)-1 mM EDTA (potassium salt). The recovered suspension was sonicated for 5 min at 3°C, divided into small portions, and stored in a vapor-phase liquid-nitrogen freezer.Choline acetyltransferase activity was assayed by a method modified (manuscript in preparation) from that of Schrier and Shuster (9). Each reaction contained the following components in a final volume of 0.05 ml, except where noted: 50 mM potassium phosphate buffer (pH 6.8), 200 mM NaCl, 1 mM EDTA (potassium salt), 2.5 mM choline iodide, 0.5% Triton X-100 (Packard), 2.2 mM ['4C]acetyl CoA (10 Ci/mol), 0.1 mM neostigmine methylsulfate, and 0-0.5 mg of homogenate protein. Each reaction was incubated at 37°C for 10 min; then 0.5 ml of H20 at 3°C was added and the diluted reaction and 2 subsequent 1.0-ml washes were passed through a 0.5 X 5 cm column of Bio-Rad AG 1-X8 resin (C1-form, 100-200 mesh). Each eluate was collected in a scintillation vial; 10 ml of scintillation solution [1000 g Triton X-100-2 liters of toluene-165 ml Liquifluor (New England Nuclear Co.)] was added and radioactivity was determined. The counting efficiency for 14C was 80-90%. ,umol of unlabeled acetylcholine, and 0.2 ,umol of unlabeled acetylcarnitine were subjected to ascending paper chromatography for 16-24 hr with 1-propanol-0.1 N acetic acid 3:1. Chromatograms were dried and sprayed with the Dragendorf reagent (18) to visualize acetylcholine or acetylcarnitine. The chromatogram was cut into 1.0 X 0.5 cm segments, and the radioactivity of each was determined with a ...
Abstract. 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.
Although the main symptoms of dementia consist of neuropsychological impairment, particularly long-term memory, dementia often involves severe behavioral and psychological symptoms of dementia (BPSD). There are quite a few patients whose BPSD are untreatable with medication. Such BPSD often have some characteristics similar to traumatic symptoms and appear related to the recollection of disturbing past traumatic events. Because the standard protocol of eye movement desensitization and reprocessing (EMDR) is not directly applicable to patients with dementia, we developed a modified protocol, the on-the-spot-EMDR method. This study describes the protocol and evaluates its application to three patients with moderate to severe dementia. Clear therapeutic effects were evident, and all three individuals showed pronounced improvement in BPSD, with results maintained at 6-month follow-up. The relevance of these findings is discussed and suggestions made for future research.
Intracellular microelectrode studies -of passive membrane properties and action potential generation were carried out on cloned and uncloned mouse neuroblastoma cells in tissue culture. The cloned cells were studied between the eighth and tenth months and the uncloned cells between the third and fifth weeks after primary dissociation. Electrophysiologic measurements of cell membrane properties were made by passing stimulating current pulses across the cell membrane from an intracellular microelectrode and recording simultaneously from the same electrode, by means of a bridge circuit, the changes in membrane potential. The range of responses to electrical stimulation varied from passive increases in membrane potential to repetitive firing of action potentials. A 20 fold range in spike generating capability was found. Passive membrane properties (membrane potential, specific membrane resistivity, and specific membrane capacitance) were similar to those of sympathetic neurons in intact preparations. Seventy-nine percent of the cloned cell line compared to 94% of the uncloned line were capable of generating action potentials. Less than 2% of the cloned cells showed repetitive firing whereas 23% of the uncloned cells had this property. As in several types of normal neurons, the action potential mechanism was largely, although not completely, blocked by iontophoretic and bath applied tetrodotoxin.
The distribution of prostaglandin D synthetase activity was determined in various tissues of rat by using the supernatant fraction (10,000 X g, 20 min) of the homogenates. The highest activity was found in brain, spinal cord, and alimentary tract. The activity was ubiquitously distributed in all parts of brain, and the highest specific activity was found in hypothalamus and thalamus. Homogenates of two neuroblastoma cell lines were found to produce prostaglandin D2, whereas a glioma cell line was almost inactive. Prostaglandin D2 is a potent and specific activator of the adenylate cyclase system of cultured neuroblastoma cells, suggesting the possibility that it may act as a neuromodulator in the central nervous system.Prostaglandin (PG) D synthetase (isomerase) was first detected in the cytosol fraction of various rat tissues (1). Recently, the enzyme was purified to homogeneity from rat brain (2) and clearly distinguished from glutathione S-transferase. To clarify the physiological role of PGD2, we carried out a systematic survey of the enzyme's distribution. In this report, we present quantitative data on the occurrence of PGD synthetase in rat tissues, particularly in brain and cultured neuroblastoma cells. Furthermore, PGD2 at about 10 nM was demonstrated to increase the cyclic AMP level rapidly in the neuroblastoma cells. The possible significance of these findings is discussed. MATERIALS AND METHODSMaterials. [1-14C]Arachidonic acid (60.2 mCi/mmol;/Ci = 3.7 X 1010 becquerels) was purchased from the Radiochemical Centre (Amersham). Arachidonic acid was a product of P-L Biochemicals. PGD2, -E2, -E1, -F2,,, -F20, and -B2, 6-keto-PGFi,, and thromboxane B2 were kindly donated by M.Hayashi (Ono Central Research Institute). L-Norepinephrine bitartrate, 3-hydroxytyramine (dopamine) HCl, DL-propranolol-HCI, 9,10-dihydroxyergotamine tartrate, and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma. Naloxone.HCI and chlorpromazine.HCI were generous gifts from Endo Laboratories and Takeda Research Laboratories (Osaka), respectively. Dithiothreitol and p-chloromercuribenzoic acid were supplied by Wako Pure Chemicals (Osaka); 1-chloro-2,4-dinitrobenzene and phenoxybenzamine.HCI were from Tokyo Kasei Kogyo (Tokyo); and precoated silica gel F254 glass plates were from E. Merck (Darmstadt). [1-'4C]PGH2 was prepared as described (2).Preparation of Tissue Homogenates. Male Wistar rats (300-350 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), perfused with 200 ml of ice-cold saline, and killed by exsanguination. Eleven organs (whole brain, spinal cord, lung, heart, liver, stomach, small intestine, kidney, spleen, adrenal gland, and vesicular gland)The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6231were quickly removed. The brain was chilled on ice and separated into 11 parts-cerebral neocortex, cerebellum, pon...
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