Tissue culture assay of Nerve Growth Factor and of the specific antiserum

Fenton, E. L.

doi:10.1016/0014-4827(70)90645-2

Cited by 166 publications

(58 citation statements)

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“…The amount of NGF was. estimated by measuring the absorbance of the preparation at 280 nm (A of 1 mg/ml = 1.6) and its activity was determined using the chick embryo dorsal root ganglion ,amy (15 The animals were killed by a blow to the head, and superior cervical ganglia were removed and decapsulated. Three pairs of ganglia were incubated in 0.5 ml of.…”

Section: Methodsmentioning

confidence: 99%

The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat.

Nikodijevic¹,

Nikodijevic²,

Yu³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Nerve grdwth factor (NGF) produces a several-fold increase in the cyclic AMP concentration in rat superior cervical ganglia in organ culture within 5 min. An increase can be seen with as little as 40 ng/ml of NGF. Oxidized NGF is without effect. The increase in the cAMP concentration produced by NGF is prevented by the addition of antiserum to NGF.Nerve growth factor (NGF) is known to be involved in the growth, development, and maintenance of sympathetic and sensory ganglia (1, 2). Administration of NGF promotes outgrowth of neuronal processes both in vivo and in vitro (3, 4). Further, there is an overall stimulation of anabolic pathways (5-7). In sympathetic neurons, there is a selective increase in the activities of tyrosine hydroxylase and dopamine f3-hydroxylase, enzymes involved in the synthesis of the transmitter norepinephrine (8). Specific receptors for NGF are present in sympathetic and sensory ganglia (9)(10)(11) and at the synaptic endings of sympathetic and sensory neurons (12, 13).However, there is no evidence concerning the intracellular events which occur between the binding of NGF to its receptors and the appearance of outgrowth or the induction of tyrouine hydroxylase. In order to obtain such information we have done experiments on the possible relationship between NGF and the concentration of the known intracellula "second messenger," cyclic 3':5'-adenosine monophosphate (cAMP). The present data show that 2.5S NGF produces a rapid and marked increase in the cAMP concentratioin in organ cultures of superior cervical ganglia from 21-day-old rats. This increase occurs in response to physiological concentrations of NGF, but not to the biologically inactive derivative, oxidized NGF. The response is prevented by antiserum from sheep immunized with NGF. MATERIALS AND METHODSNGF was prepared in the 2.5S form from the salivary glands of mature male mice by the method of Bocchini and Angeletti (14) and stored frozen in small portions. The amount of NGF was. estimated by measuring the absorbance of the preparation at 280 nm (A of 1 mg/ml = 1.6) and its activity was determined using the chick embryo dorsal root ganglion ,amy (15 The animals were killed by a blow to the head, and superior cervical ganglia were removed and decapsulated. Three pairs of ganglia were incubated in 0.5 ml of. BGJ enriched media (Fitton-Jackson Modification, Grand Island Biological Co.) for 60 min at 370 in an atmosphere of 95% 02 and 5% CO2 before the addition of NGF or of the appropriate control buffer.For cAMP assay the ganglia were rinsed in ice-cold 0.25 M sucrose for 2 min, blotted on filter paper, and homogenized in 0.6 ml of ice-cold 5% trichloroacetic acid. After 15 min the homogenate was centrifuged and the supernatant portion was washed three times with 2 volumes of ether. The aqueous phase was assayed in quadruplicate for cAMP by the competitive binding assay described by Gilman (16), using kits provided by Amersham-Searle. Each experiment included an identical set of duplicate samples with an internal standar...

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Section: Methodsmentioning

confidence: 99%

The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat.

Nikodijevic¹,

Nikodijevic²,

Yu³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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“…The NGF content of heart ventricle and skeletal muscle was too low to be measured (<0.3 ng/g wet weight), and no NGF was found in the serum (<0.05 ng/ml). For SCG, the tissue with the highest NGF concentration measured by EIA, estimation of NGF content was also possible by the chicken dorsal root ganglia bioassay (20). However, because concentrated homogenates had to be used, the evaluation was difficult (cloudiness of the plasma clot) and consequently the scattering of the biological response was high, ranging between 0.002 and 0.01 biological unit per SCG, corresponding to 10-70 pg of NGF per SCG.…”

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confidence: 99%

“…One antibody-coated glass bead per well of a microtiter plate (Greiner, Nfirtingen, Federal Republic of Germany) was incubated overnight at room temperature with 50 /.l of sample or mNGF standard solution ( Determination of Biological Activity of NGF. The biological activity was estimated by using the plasma clot bioassay of Fenton (20). Ten to 15 superior cervical ganglia (SCG) were homogenized in 150 ,ul of Dulbecco's modified Eagle medium containing 10% heat-inactivated fetal calf serum, 2% bovine serum albumin, 2% gelatin, 0.1 mM leupeptin, aprotinin at 20 kallikrein units/ml, penicillin at 200 units/ml, and streptomycin at 200 units/ml.…”

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confidence: 99%

Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation.

Korsching¹,

Thoenen²

1983

Proc. Natl. Acad. Sci. U.S.A.

468

247

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A two-site enzyme immunoassay is described which does not suffer from artifacts inherent in previous assays and has the necessary high sensitivity to determine the endogenous levels of nerve growth factor (NGF) in the sympathetic nervous system and its target organs. Monoclonal and affinity-purified polyclonal antibodies against mouse NGF (mNGF) were covalently linked to glass beads as the first site and coupled to the enzyme (t-galactosidase as the second site. Detection of the fluorescent 13-galactosidase reaction product permitted the determination of 0.01-0.02 fmol of mNGF per assay. The recovery of mNGF added to homogenates varied between 50% and 100%, depending on the tissue. Rat superior cervical and stellate ganglia were found to contain (mean ± SEM) 25 ± 4 and 19 ± 3 ng of NGF per g wet weight, respectively, and the densely innervated submandibular gland, heart atrium, and iris contained 0.5 ± 0.1, 1.0 ± 0.1, and 1.9 ± 0.3 ng of NGF per g wet weight, respectively. Heart ventricle and skeletal muscle, which are poorly innervated by the sympathetic nervous system, did not contain detectable levels of NGF (<0.3 ng/g wet weight). Serum contained <0.05 ng of NGF per ml. The correlation between NGF levels and density of innervation is consistent with the concept that the production of NGF in target organs determines their density of innervation by the sympathetic nervous system. Nerve growth factor (NGF) is a protein essential for the development and maintenance of function of the peripheral sympathetic nervous system (for a review, see ref. 1). However, evidence of the local synthesis of NGF in sympathetically innervated effector organs and of its function as a retrograde trophic messenger between effector organs and innervating neurons has only been indirect. (a) Exogenous NGF is taken up by a selective and saturable mechanism by sympathetic nerve terminals and is then transported retrogradely to the corresponding perikarya, where it evokes its characteristic biochemical effects (1, 2). (b) Interruption of the retrograde axonal transport by surgical or chemical procedures has the same effect as the neutralization of endogenous NGF by specific antibodies (1-3). Thus, during early stages of development, both interruption of retrograde axonal transport and neutralization of endogenous NGF by antibodies lead to a degeneration of the corresponding neurons. In differentiated neurons, the two procedures lead only to an impairment of function reflected by a decrease in levels of enzymes involved in the synthesis of the adrenergic transmitter norepinephrine.Despite many attempts, it has not been possible to determine the levels of NGF in sympathetically innervated target tissues by reliable quantitative methods (4, 5). With bioassay techniques, NGF became detectable only if the tissues were denervated either in situ (6) or by being brought into culture (6, 7). This suggests that the levels of NGF in sympathetically innervated target tissues are extremely low because the locally synthesized NGF is rapidly re...

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“…Antibodies against 2.5 S NGF were raised in goats and purified by affinity chromatography according to Stockel, Gagnon, Guroff & Thoenen (1976). The biological activity of the antibodies was tested by the inhibition of the NGF-mediated fibre outgrowth in the dorsal root ganglion (Fenton, 1970), in which 30 ng of purified NGF-antibodies inhibited the effect of 1 ng of 2.5 S NGF. Biologically inactive NGF was obtained by oxidation of the tryptophan residues with N-bromosuccinimide as described by Frazier, Angeletti, Sherman & Bradshaw (1973).…”

Section: Methodsmentioning

confidence: 99%

“…The biological activity was 250 biological units/pg of protein, as determined by the bioassay method of Fenton (1970), using dorsal root ganglia of 10 dayold chicken embryos. The purity of standard 2.5 S NGF was assessed by 15% polyacrylamide sodium dodecyl gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Shape Changes Induced by Biologically Active Peptides and Nerve Growth Factor in Blood Platelets of Rabbits

Gudat

Laubscher

Otten

et al. 1981

British J Pharmacology

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Nerve growth factor (NGF), substance P (SP) and thymopoietin all caused shape change reactions of rapid onset in rabbit platelets. NGF had the highest maximal effect, and SP the lowest EC50 (concentration causing half maximal shape change). The action of SP was reversible within 5 min, whereas that of NGF lasted for at least 1 h. A series of other peptides were inactive. After preincubation of platelets with SP, a second application of SP no longer caused a shape change reaction, whereas the effect of NGF was not influenced. An oxidized NGF‐derivative without biological activity did not cause a shape change reaction, neither did epidermal growth factor. Prostaglandin E1 (PGE1) and pretreatment of the platelets with 3% butanol, which counteract the shape changes caused by 5‐hydroxytryptamine (5‐HT) and adenosine 3′, 5′‐diphosphate, also antagonized those induced by NGF and SP. Neither heparin nor methysergide, an antagonist of 5‐HT‐receptors, influenced the shape change induced by NGF or SP. The action of NGF was also antagonized by a specific antibody to NGF. Thymopoietin, like the basic polypeptide polyornithine (mol. wt. 40,000), was not antagonized by PGE1 and butanol. Heparin, which counteracted the effect of polyornithine, did not influence that of thymopoietin. In conclusion, different modes of action are involved in the shape change of blood platelets induced by polypeptides and proteins. SP and NGF may act by stimulating specific membrane receptors.

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Tissue culture assay of Nerve Growth Factor and of the specific antiserum

Cited by 166 publications

References 5 publications

The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat.

The effect of nerve growth factor on cycle AMP levels in superior cerival ganglia of the rat.

Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation.

Shape Changes Induced by Biologically Active Peptides and Nerve Growth Factor in Blood Platelets of Rabbits

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