Albee Messing

Transgenic mice harboring SV40 t-antigen genes develop characteristic brain tumors

A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice.

Cholinergic agonist-induced down regulation of neuronal α-bungarotoxin receptors

Abstract The ability of cholinergic ligands to regulate neuronal α-bungarotoxin receptor number was studied in dissociated monolayer cultures of embryonic chick ciliary gangliion neurons. Carbamylcholine and nicotine, but not D -tubocurarine, caused a loss of 25% of the surface [125I]α-bungarotoxin receptors within 1 h at 37°C. This receptor loss occured withour change in affinity for [125I]α-bungarotoxin, was temperature-sensitive and was prevented by co-incubation with D -tubocurarine.

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons.

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.

Extra-synaptic localization of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

Surface [125I]alpha-bungarotoxin receptors were localized on dissociated cultures of embryonic chick ciliary ganglion neurons by electron microscopic autoradiography. Grain density was 4-5-fold higher on cell body membrane versus neuritic process membrane, and there was no apparent concentration of grains at morphologically identifiable inter-neuronal synapses.

Developments of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

Abstract We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [ 125 I]α-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 μg/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [ 125 I]α-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [ 125 I]α-bungarotoxin was saturable with respect to both time of incubation (20–30 min) and concentration of toxin (5–10 nM), with an apparent K d = 1.0 nM. Binding sites for [ 125 I]α-bungarotoxin increased during the first week in culture from 1.8 fmol per 10 4 neurons at 1 day in vitro (DIV) to 8.6 fmol per 10 4 neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of α-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal α-bungarotoxin receptors.