Regulation of nerve growth factor receptor expression on Schwann cells.
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Aging↗
Biomedical subjects
E M Johnson
Publications and source records attributed to E M Johnson.
Identification of a truncated form of the nerve growth factor receptor.
Schwann cells express growth factor (NGF) receptors on their cell surface in response to axotomy, a phenomenon that can be demonstrated both in vivo and in vitro. The predominant form of the NGF receptor on Schwann cells exists as an approximately equal to 80-kDa band, as determined by NaDodSO4/PAGE. We demonstrate that cultured Schwann cells shed a truncated (50-kDa) form of the NGF receptor (NGF-Rt) into their medium. Other cell types that shed the NGF-Rt into medium include a rat schwannoma and, to a lesser extent, PC12 cells and superior cervical ganglion neurons. NGF-Rt was not found in media conditioned by mixed neuron/glia cultures from various brain regions, or anterior pituitary cells derived from rat. In vivo, NGF-Rt was present in neonatal rat urine, and its presence was developmentally regulated: levels were high in postnatal day-1 rat urine and declined to low, but detectable, levels by weeks 4 and 8. NGF-Rt was also found in amniotic fluid and in the stomach contents of fetal rats. Maternal urine (pre- and postnatal) had slightly elevated NGF-Rt levels over normal adult urine. NGF-Rt was detected in rat plasma and showed developmental regulation similar to that found for urine. In addition, a 77-kDa receptor species was detected in plasma during early development. Finally, NGF-Rt was significantly elevated in the urine of adult rats with bilateral sciatic nerve lesions. These findings suggest that the developmentally regulated release of NGF-Rt, present in plasma and other body fluids, plays a regulatory role in nervous system development.
Aging↗
Asbestos fibers mediate transformation of monkey cells by exogenous plasmid DNA.
We have tested the ability of chrysotile asbestos fibers to introduce plasmid DNA into monkey COS-7 cells and the ability of this DNA to function in both replication and gene expression. Chrysotile fibers are at least as effective as calcium phosphate in standard transfection assays at optimal ratios of asbestos to DNA. After transfection with chrysotile, a minor percentage of introduced plasmid DNA bearing a simian virus 40 origin of replication replicates after 24 hr. Fragmentation of entering DNA is more prominent with asbestos than with calcium phosphate, and after 72 hr most DNA introduced by asbestos is associated with chromosomal DNA. Cells transfected with plasmid p11-4, bearing the p53 protooncogene, express this gene. Cells transfected with pSV2-neo express a gene conferring resistance of antibiotic G418, allowing isolation of colonies of transformed cells after 18 days. The introduction of exogenous DNA into eukaryotic cells could cause mutations in several ways and thus contribute to asbestos-induced oncogenesis.
Animals↗
Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.
We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.
Animals↗
Postnatal effects of prenatal insult.
Exogenous agents may perturb development during the embryonic period and adversely affect the formation of organs. However, adverse effects on development are not limited to the embryonic period nor are the manifestations restricted solely to outright gross structural malformation, but may instead be expressed as a decrement or abberration of postnatal function. Susceptibility to altered development may extend well into the postnatal period. Studies of functional parameters in several organ systems have demonstrated the broad-based susceptibility, subtlety of expression and potential of long-lasting effects of altered development assessed by physiologic assays. Adverse effects on functional development, whether in the CNS, reproductive, gastrointestinal, genitourinary, respiratory, or immune systems, etc., merit continuing investigation. From the viewpoint of risk estimation and hazard detection, evaluations of postnatal functional parameters may be relevant for several reasons. First, such parameters may serve as low-dose triggers. Second, they may be useful as a focal point for epidemiological studies. Finally, a more thorough understanding of the degree and magnitude of such postnatal functional deficits is needed since an adverse maternal effect may be transient, considered acceptable, or unperceived, but the effect on the conceptus may be permanent and severe. The immune and respiratory systems are discussed as two examples of how subtle and protean adverse effects on functional development may be.
Animals↗
Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro.
Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.
Acyclovir↗
Nerve growth factor receptors on cultured rat Schwann cells.
Neonatal rat Schwann cells were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 125I-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not saturable at high ligand concentrations (50-70 nM) and that a high-capacity, low-affinity NGF binding component existed on these cells as compared to PC12 cells. The monoclonal antibody, 192-IgG, which recognizes the rat NGF receptor, was used as an immunohistochemical tool to verify the presence of NGF receptors on the cultured rat Schwann cells. In contrast to radiolabeled NGF, 125I-192-IgG demonstrated saturable binding to Schwann cells in suspension, with Kd and Bmax values of 4 nM and 115 fmol/10(6) cells, respectively. Schwann cells showed no evidence of slow dissociation or internalization of NGF binding at any of several NGF concentrations. 192-IgG was used to immunoprecipitate 125I-NGF chemically crosslinked to cell membranes. SDS-PAGE and subsequent autoradiography of the immunoprecipitated NGF receptors revealed that 2 species of NGF receptors were precipitated from Schwann cells and PC12 cells. In PC12 cells, 2 bands with molecular weights of 90 and 210 kDa were identified. The Schwann cell NGF receptor species migrated slower on the gels, with apparent molecular weights of 95 and 220 kDa. Further analysis of glial cell NGF receptors showed that Schwann cells isolated from the vagus nerve of neonatal rats also expressed NGF receptors in culture; however, astrocytes cultured from neonatal rat cerebral cortex, cultured under conditions reported here, were devoid of detectable NGF receptors. These results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann cells in vivo when adult peripheral nerve is injured. The data are discussed in terms of a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.
Animals↗
Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties.
Axotomy of sciatic nerve fibers in adult rats induces expression of NGF receptor in the entire population of Schwann cells located distal to the injury (Taniuchi et al., 1986b). In the present study we have used immunocytochemistry, with a monoclonal antibody directed against the rat NGF receptor, to examine axotomized peripheral nerves by light and electron microscopy. We have found that (1) the NGF receptor molecules were localized to the cell surface of Schwann cells forming bands of Bungner; (2) axonal regeneration into the distal portion of sciatic nerve coincided temporally and spatially with a decrease in Schwann cell expression of NGF receptor; (3) Schwann cell NGF receptor could be induced by axotomy of NGF-independent neurons, such as motoneurons and parasympathetic neurons; and (4) the presence of axon-Schwann cell contact was inversely related to expression of Schwann cell NGF receptor. Using biochemical assays we have found that, in striking contrast to peripheral nerves, there was no detectable induction of NGF receptor in the spinal cord and brain after axotomy of NGF receptor-bearing fibers. Filtration assays of 125I-NGF binding to the induced NGF receptors of Schwann cells measured a Kd of 1.5 nM and a fast dissociation rate, both characteristics of class II receptor sites. We conclude that Wallerian degeneration induces Schwann cells, but not central neuroglia, to produce and position upon their plasmalemmal surface the class II NGF receptor molecules. The induction is ubiquitous among Schwann cells, irrespective of the type of axon they originally ensheathed. Expression of Schwann cell NGF receptor is negatively regulated by axonal contact, being induced when axons degenerate and suppressed when regenerating axons grow out along the Schwann cell surface. We propose that the induced NGF receptors function to bind NGF molecules upon the Schwann cell surface and thereby provide a substratum laden with trophic support and chemotactic guidance for regenerating sensory and sympathetic neurons.
Animals↗
An immunohistochemical study of the nerve growth factor receptor in developing rats.
Nerve growth factor (NGF) receptor expression was studied in rats between embryonic day 11 (E11) to postnatal day 10 (PND10) by using a monoclonal antibody, 192-IgG, that specifically recognizes rat NGF receptor. Sympathetic ganglia were lightly stained by 192-IgG for NGF receptor immunoreactivity (NGFRI) (E11-PND10). Neural crest-derived sensory ganglia were moderately to densely stained (E11-PND10). Areas in CNS innervated by the central processes of these ganglia were also stained. Parasympathetic ciliary ganglion showed some detectable staining (E16-PND6). Placode-derived sensory ganglia were stained more densely than that of neural crest-derived sensory ganglia. The most densely stained tissue for NGFRI was found in all peripheral nerves. Basal forebrain cholinergic neurons were NGFRI positive from E15 throughout the period examined. Motoneurons in both spinal cord and brain stem were positive for NGFRI between E15 and PND10. NGFRI staining was seen in a variety of sensory pathways and related structures, such as olfactory tract and glomerular layer of olfactory bulb; retina, optic nerve and tract, lateral geniculate nucleus, medial terminal nucleus of the accessory optic tract, and olivary pretectal nucleus; ventral cochlear nucleus and to a lesser degree in dorsal cochlear nucleus, superior olive, and nucleus of lateral lemniscus; solitary tract; cuneate nucleus, gracile nucleus, and ventroposterior thalamic nucleus. The specific staining was also found in some other CNS structures, including brain-stem reticular formation; amygdala; medial nucleus of inferior olive but not the rest of inferior olive, external granule cell layer and Purkinje's cells of cerebellum, and deep cerebellar nuclei. Some non-neuronal tissues such as meninges and dental tissue showed very distinctive staining. Limb buds and somites were NGFRI positive starting at E11, and the staining on muscle tissue became very dense at E15-E18 and largely disappeared around PND10. Embryonic thymus was positive for NGFRI. The adventitia surrounding blood vessels was very densely stained. The changes in NGFRI staining seen in this study suggest that NGF may have broader effects during development than previously thought.
Animals↗
Rapid DNA analysis of alpha 1-antitrypsin deficiency: application of an improved method for amplifying mutated gene sequences.
The gene coding for alpha 1-antitrypsin (AAT) is mutated in some individuals to code for a protein, the PiZ variant, associated with alpha 1-antitrypsin serum deficiency. Homozygotes for this defect are at high risk for various liver diseases and for early onset emphysema. Here, we employ an enzymatic amplification procedure for diagnosis of a Z-specific gene mutation in cellular DNA, and we describe a new procedure for selective enhancement of the mutated sequence. We have synthesized 2 oligonucleotides which prime amplification of a 156 bp region of exon V including a single base mutation specific for the PiZ variant. Amplification of 2 to 6 X 10(5)-fold is obtained allowing rapid detection of the Z mutation, using selective oligonucleotide probes, in unseparated genomic DNA of cells from family members homozygous or heterozygous for the defect. New methodology allows enzymatic amplification at elevated temperatures. To take advantage of this, we have developed a method using discriminating oligonucleotides as primers that allows enhanced sensitivity of hybridization. These methods allow diagnosis of the Z form of AAT deficiency within a single day using submicrogram quantities of DNA.
Base Sequence↗
Nerve growth factor (NGF) receptors in the central nervous system.
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Animals↗
Human infection with Ehrlichia canis.
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Ehrlichia↗
Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms.
Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo[2-tritium]acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription.
Acetylation↗
Partial purification of a parasympathetic neurotrophic factor in pig lung.
Previous work has shown that pig lung, among a number of tissues examined, contained relatively high amounts of survival-promoting and choline acetyltransferase (ChAT)-sustaining activities for parasympathetic, ciliary ganglion neurons from chicken embryos. It is reported here that these two activities have been partially purified and shown to coelute from several chromatographic columns, indicating that the activities probably reside in the same molecule. Three species were detected with apparent molecular weights (MW) of over 100 kDa, 40 kDa, and 3 kDa, and the two high-MW species converted into the low-MW species. The high-MW species were associated with neuritic outgrowth, whereas the low-MW species was not. This low-MW form was found to maintain survival and ChAT activity, whose EC50s were closely correlated, and was partially purified 3400-fold. The partially purified material had an EC50 of 550 ng protein/ml, and was heat-stable. It was resistant to deoxyribonucleases, ribonucleases, proteases, and neuraminidase. As previously found with the crude fraction of pig lung, the trophic effects were specific for parasympathetic neurons; neither sympathetic nor peripheral sensory neurons were affected. These findings indicate that the pig lung factor(s) has a more specific target spectrum than previously reported ciliary neuron survival factors, suggesting that it may be a novel and important neurotrophic substance.
Animals↗
Sympathetic neuronal destruction in macaque monkeys by guanethidine and guanacline.
To determine whether the peripheral sympathetic neurons of subhuman primates are destroyed by guanacline treatment, we treated Macaca fasicularis with 2 or 20 mg/kg of guanethidine, guanacline, or the saturated analog of guanacline (SAG) 5 times per week for 4 or 12 weeks. All monkeys given 20 mg/kg of guanethidine, guanacline, or SAG showed a marked loss of neurons in the ganglia of the peripheral sympathetic nervous system. Treatment of macaques with 2 mg/kg of the guanidinium compounds resulted in patches of small-cell infiltrate, slight neuronal loss, and degenerative alterations in the sympathetic ganglia. Neuronal alterations in sympathetic ganglia of all treated monkeys were accompanied by a prominent heterogeneous infiltrate of mononuclear cells arranged primarily in a perivascular distribution and extending into the ganglionic neuropil. Peripheral sensory ganglia were unaffected. These histological findings are similar to those described in the guanethidine-induced immune-mediated sympathectomy, which has been extensively studied in the rat.
Animals↗
Use of the adult developmental relationship in prescreening for developmental hazards.
The overwhelming majority of chemicals already in commerce or brought into use each year have not been evaluated for their potential to adversely affect in utero development. Data from those that have been evaluated thus far in pregnant laboratory animals establish that most, but not all, were no more hazardous to the conceptus than they were to adult homeostasis. Most did not need standard developmental toxicity testing because avoidance of adult toxic exposure levels would have precluded abnormal in utero development. The six general principles of teratology when modified, expanded, and placed into this type of context of contemporary developmental toxicology allow an updating of the present testing sequence which was devised prior to 1966. The developmental hazard index (A/D ratio) calculated from the adult and developmental NOELs of standard Segment II evaluations is predicted by in vitro means. This determination, when coupled with adequate considerations of exposure can be used to prioritize chemicals for more elaborate developmental toxicity tests. Those chemicals with large ratios, i.e., disruptive of embryogenesis at treatment levels too low to produce overt effects in the mother and/or with significant concern regarding exposure, can be identified and tested in pregnant laboratory animals as high priority items. Those with low ratios and those for which there is a low level of concern regarding exposure potential also can be identified and are not high priority items for testing in pregnant animals. The proposed tier system establishes priorities of testing based on exposure and the concept of target organ toxicity applied to the embryo. It provides intensive in vivo evaluations of those chemicals for which developmental effects testing is most needed and avoids use of resources and animals for unnecessary testing of agents that do not pose threats to the conceptus.
Animals↗
Consensus workshop on the evaluation of maternal and developmental toxicity work group I report: end points of maternal and developmental toxicity.
It was concluded that this conference, sponsored by the EPA, has provided an opportunity to state current ideas regarding the utility of data from studies in developmental toxicology and that the deliberations have highlighted the need to consider fundamental relationships between maternal and developmental effects in a more comprehensive manner in the future.
Animals↗
A tier system for developmental toxicity evaluations based on considerations of exposure and effect relationships.
The backlog of untested chemicals and the rate at which new substances enter the marketplace exceed our capacity for developmental effects testing by standard in vivo methods. However, conservative use of two observations in a manner consistent with present day understanding of abnormal developmental biology can more accurately focus attention and resources on those agents in greatest need of complex testing for effects on in utero development. These two observations are 1) most chemicals are no more toxic to embryonic development than they are to adult homeostasis and 2) most human exposure to chemicals is de minimus, i.e., so small that it is inconsequential. Recently devised in vitro assays to quantitatively rank chemicals according to their developmental hazard index, when used in conjunction with more conventional in vivo methods and appropriate considerations of exposure, permit evaluation of a significantly larger number of chemicals than is currently achieved. The methods described apply a tier approach to establish testing priorities that markedly reduce the time, cost, and number of laboratory animals needed for evaluation of developmental toxicity.
Abnormalities, Drug-Induced↗