Biomedical subjects
Y A Barde
Publications and source records attributed to Y A Barde.
Purification of a new neurotrophic factor from mammalian brain.
We report the purification from pig brain of a factor supporting the survival of, and fibre outgrowth from, cultured embryonic chick sensory neurons. The purified factor migrates as one single band, mol. wt. 12 300, on gel electrophoresis in the presence of sodium dodecylsulphate (SDS) and is a basic molecule (pI greater than or equal to 10.1). Approximately 1 microgram factor was isolated from 1.5 kg brain. The final degree of purification was estimated to be 1.4 X 10(6)-fold, and the specific activity 0.4 ng/ml/unit, which is similar to that of nerve growth factor (NGF) using the same assay system. This factor is the first neurotrophic factor to be purified since NGF, from which it is clearly distinguished because it has different antigenic and functional properties.
Presence and disappearance of nerve growth factor receptors on sensory neurons in culture.
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Extract from brain stimulates neurite outgrowth from fetal rat retinal explants.
Explants from rat fetal retina were placed in culture and assayed for fiber outgrowth. In contrast to results obtained with lower vertebrates, nerve growth factor (NGF) does not seem to play a role in this system: NGF is not able to stimulate fiber outgrowth and antibodies to NGF do not block the spontaneously occurring fiber outgrowth. However, an extract prepared from pig brain is able to stimulate fiber outgrowth in a dose-dependent manner. It is suggested that such an extract can be used as a source of putative neurotrophic factors exhibiting in the mammalian central nervous system (CNS) an action similar to that of NGF in the peripheral nervous system (PNS) of mammals and in the CNS of lower vertebrates like fishes and amphibia.
Subpopulations of cultured chick sympathetic neurones differ in their requirements for survival factors.
Peripheral autonomic and sensory neurones derived from the neural crest will survive in vitro only if the culture medium is supplemented with specific factors (for review see ref. 1). For example, the nerve growth factor (NGF), although supporting the survival of sympathetic and spinal sensory neurons, is ineffective on parasympathetic neurones, whereas medium conditioned by chick heart cells (HCM) supports the survival of all three neuronal types. We showed previously that the requirements of cultured spinal sensory ganglion neurones for survival factors changed during development, and so provided a basis for the classification of such factors. We now demonstrate that cultured post-mitotic neurones from chick paravertebral sympathetic ganglia respond differentially to NGF, HCM and medium conditioned by C6 glioma cells (GCM). Thus, there three survival factors are functionally distinct in that they support the survival in culture of discrete subpopulations of sympathetic neurones. Those subpopulations responding to HCM and NGF are shown to differ not only in their requirements for survival factors but also in their contents of the cholinergic and adrenergic marker enzymes choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH).
Production of specific antibodies to choline acetyltransferase purified from pig brain.
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The role of nerve growth factor (NGF) and related factors for the survival of peripheral neurons.
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Sensory neurons in culture: changing requirements for survival factors during embryonic development.
The effect of nerve growth factor (NGF) and medium conditioned by glioma cells (GCM) on the survival of chicken sensory neurons in culture was investigated. Neurons were isolated from embryos 8 days (E8) to 16 days (E16) old and the proportion of surviving neurons was determined after 2 days in culture. In the absence of NGF or GCM, essentially no neurons survived at any age. In the presence of NGF, survival increased from 25% of the neurons at E8 to 40% between E10 and E12 and then decreased to background level (5%) at E16. In contrast, in the presence of GCM, survival increased continuously from 10% of the neurons at E8 to 75% at E16. At early developmental stages, the effect of NGF and GCM together was greater than the sum of their individual effects: at E8, about 80% of the neurons survived, double the number expected for a simple additive effect. Thus, a significant proportion of chicken neurons from dorsal root ganglia require both NGF and GCM for survival, and this may well include neurons from the ventro-lateral population, which do not respond to NGF alone. As neurons matured, the double requirement progressively decreased and, by E16, NGF no longer increased the number of neurons over that surviving in response to GCM alone. The facts that rat brain extracts mimicked the effect of GCM and that the potency of the brain extracts of rat in the postnatal period increased in parallel with the development of the glial cells suggest that glial cells produce a factor(s) both immunologically and functionally different from NGF which supports the survival of sensory neurons.
Physiology of nerve growth factor.
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Guinea pig prostate is a rich source of nerve growth factor.
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Induction of fibre outgrowth and choline acetyltransferase in PC12 pheochromocytoma cells by conditioned media from glial cells and organ extracts.
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Mechanism of action and possible sites of synthesis of nerve growth factor.
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New factor released by cultured glioma cells supporting survival and growth of sensory neurones.
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Nerve growth factor in mouse and rat serum: correlation between bioassay and radioimmunoassay determinations.
High levels of nerve growth factor (NGF) determined by competition radioimmunoassay do not agree with values obtained by bioassay. This discrepancy is illustrated here with rat and mouse serum as examples in which values up to 1000 ng/ml have been found by using competition radioimmunoassays. An explanation for the discordant results is presented: serum components bind NGF with an intermediate affinity (Kd = 10(-7) M) but with a very large capacity (up to 0.5 mg of NGF per ml of rat serum). The binding of 125I-labeled NGF to serum components competes with the binding to the solid-phase antibodies (Kd = 10(-9)M) present in limiting amounts, according to the principle of competition radioimmunoassays. Thus, less radioactivity is recovered bound to the antibodies and this gives the erroneous impression that NGF is present. To overcome this difficulty, a two-site radioimmunoassay has been developed which utilizes nonlimiting numbers of antibody binding sites. This assay provides a reliable determination of NGF levels in serum and it can be shown that in rat and mouse serum (either sex) there is less than 5 ng of NGF per ml, in agreement with the results of the bioassay.
Potassium-induced increase in oxygen consumption of brown adipose tissue from the rat.
1. In brown adipose tissue, noradrenaline induces an increase in respiration and a depolarization of the cells. The effect of an increase in potassium concentration in a range known to depolarize the brown adipocytes was tested on the O2 consumption. 2. Isolated interscapular brown adipose tissue from the rat was incubated in chambers that allowed O2 consumption to be measured over prolonged periods. 3. 45-50 mM-KC1 were found to induce a more that fourfold increase in O2 consumption, which was stable, reversible and dependent upon the presence of calcium in the meduim. 4. When rats were pre-treated with reserpine or 6-hydroxydopamine the KC1-induced increase in O2 consumption was sharply reduced or entirely adsent. 5. The effect of KC1 was greatly inhibited by (-)-propranolol, but not by (+)-propranolol. 6. Moderate increases in O2 consumption induced by low concentrations of potassium were potentiated by desipramine, a drug which is known to block the uptake of catecholamines by adrenergic nerve endings. 7. Surgical denervation caused a decrease in the catecholamine content of the tissue, but had no effect on the KC1 response. 8. It is concluded that in brown adipose tissue, potassium stimulates O2 consumption by causing a release of noradrenaline from nerve endings. This implies that surgical denervation as it is commonly performed on this tissue does not denervate the brown adipocytes but probably only the blood vessels.
Different factors from the central nervous system and periphery regulate the survival of sensory neurones.
Work on nerve growth factor has established that the survival of developing vertebrate neurones depends on the supply of a neurotrophic factor from their target field. The discovery of several new neurotrophic factors has raised the possibility that neurones which innervate multiple target fields require several different neurotrophic factors for survival. Here we show that two distinct neurotrophic factors, one in the central nervous system (CNS) and the other in skeletal muscle, promote the survival of proprioceptive neurones in culture. At saturating concentrations, either factor alone supported most neurones and there was no additional survival in the presence of both factors, but at subsaturating concentrations the combined effect was additive. The neurotrophic activity of each factor was greatest during the period of natural neuronal death. Our results demonstrate that each cultured proprioceptive neurone responds to two distinct neurotrophic factors present in its respective central and peripheral target fields, and suggest that these factors cooperate in regulating survival during development.
Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section.
Motoneurons innervating the skeletal musculature were among the first neurons shown to require the presence of their target cells to develop appropriately. But the characterization of molecules allowing motoneuron survival has been difficult. Ciliary neurotrophic factor prevents the death of motoneurons, but its gene is not expressed during development. Although the presence of a neurotrophin receptor on developing motoneurons has suggested a role for neurotrophins, none could be shown to promote motoneuron survival in vitro. We report here that brain-derived neurotrophic factor can prevent the death of axotomized motoneurons in newborn rats, suggesting a role for this neurotrophin for motoneuron survival in vivo.