Electrophoretic similarity of the ciliary ganglion survival factors from different tissues and species

doi:10.1016/0012-1606(85)90367-7

Developmental Biology

Volume 109, Issue 1, May 1985, Pages 255-258

https://doi.org/10.1016/0012-1606(85)90367-7 Get rights and content

Abstract

The survival of dissociated ciliary ganglion neurons is promoted by extracts of several different embryonic and adult tissues from two species. The survival-promoting activity in each of these extracts survives exposure to sodium dodecyl sulfate (SDS) and sulfhydryl-reducing agent and can, therefore, be subjected to SDS-polyacrylamide gel electrophoresis. Upon electrophoresis, the survival-promoting activity is recovered in a discrete peak at an apparent molecular weight of approximately 21,800 for all of the tissues examined. These results suggest that a similar molecule in each of these different tissues and species may be responsible for their ability to promote the survival of ciliary ganglion nerve cells in culture.

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Cited by (11)

Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli
1991, BBA - Gene Structure and Expression
The gene ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21 (DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/1 per A₆₀₉ unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn²⁺-affinity chromatography. Parified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A₂₇₉^1% = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. Highly-purified recombinant human CNTF supported the survival of thick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.
The role of dihydropyridine-sensitive voltage-gated calcium channels in potassium-mediated neuronal survival
1989, Brain Research
The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED₅₀ = 20–25mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED₅₀ = 10.8 ± 7.0mM). In contrast, chemically closely related dihydropyridines, PN200-110 (ED₅₀ = 0.33 ± 0.15nM) and nitrendipine (ED₅₀ = 1.3 ± 0.3nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED₅₀ = 0.78 ± 0.38 μM), the benzothiazepine diltiazem (ED₅₀ = 1.7 μM), and the inorganic ion cadmium (ED₅₀ = 5.8 μM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve growth factor, basic fibroblast growth factor, or ciliary neurotrophic factor, also suggesting that voltage-gated calcium channels are not involved in the action of these factors. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.
Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells
1989, Developmental Biology
Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, ∼5% of the cells are antigen-positive (A⁺). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A⁺. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A⁺ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A⁺ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10–15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K⁺, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became “neuron-like” within 10 days. No neuron-like cells were found in liver-, notocord-, or neural tube-conditioned media if FBS was used. When A⁺ cells were eliminated either by complement-mediated cytotoxicity or by laser-ablating A⁺ cells during no-flow cytometry, all ChAT activity was also eliminated, and no neuron-like cells or ChAT activity was found in cultures during a subsequent 3-week culture period. We conclude that culture conditions that normally promote ciliary ganglion neuron survival and cholinergic development also promote differentiation and development of cholinergic neuron-like cells from the isolated A⁺ neural crest subpopulation. These results are consistent with the hypothesis that the A⁺ subpopulation contributes to the formation of ciliary ganglion neurons in the embryo.
Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium
1988, Developmental Brain Research
Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM, indicating that this early, initial responsiveness to target-released materials within this system is species-specific.
An examination of ciliary neuronotrophic factors from avian and rodent tissue extracts using a blot and culture technique
1987, Developmental Brain Research
We have previously reported a technique for determining the apparent molecular weight (M_r) of ciliary neuronotrophic factors (CNTFs) in crude extracts. This method involves SDS-polyacrylamide gel electrophoresis of the extract, Western blotting and culture of purified ciliary ganglion neurons on the paper containing the blotted lane. Neurons will survive only if in direct contact with the trophic factor band and the surviving neurons, when stained with a vital dye, will outline the CNTF band thereby indicating the M_r of the active polypeptide. Here we have modified this ‘blot and culture’ technique by (1) including M_r standard proteins in the same electrophoretic lane with the samples, (2) identifying the proteins by staining the nitrocellulose blot with Amido black, (3) marking the standard bands with pinholes and (4) destaining the blot prior to seeding neurons onto it. The active CNTF polypeptides can then be identified by their ability to support the 24-h survival of cultured ciliary neurons. This modified technique was used to determine the M_r of CNTF activities in several chick and rat tissue extracts of selected developmental ages and to ascertain if the two forms of CNTF are exclusive to chick and rat, embryonic and adult, or eye and nerve tissues. We report that (1) the above modifications permitted a more accurate method for M_r determination than the previous method, (2) only two apparent forms of CNTF were recognized, (3) the M_r found for each form is 25 kDa and 28 kDa, (4) both forms can be present in chick and rat tissues and from embryonic and adult sources and (5) the 28 kDa form is predominant in rat while the 25-kDa form is predominant in chicken tissues.
Reciprocal regulation of ciliary neurotrophic factor receptors and acetylcholine receptors during synaptogenesis in embryonic chick atria
1998, Journal of Neuroscience

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Brief noteElectrophoretic similarity of the ciliary ganglion survival factors from different tissues and species

Abstract

Brain Res

Neurosci. Lett

Anal. Biochem

J. Biol. Chem

Dev. Biol

Neurosci. Lett

Brain Res

Dev. Biol

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Cholinergic neuronotrophic factors: Intraocular distribution of trophic activity for ciliary neurons

Science (Washington, D. C.)

Human platelet-derived growth factor (PDGF): Purification of PDGF-I and PDGF-II and separation of their reduced subunits

Brief note
Electrophoretic similarity of the ciliary ganglion survival factors from different tissues and species