Search PubMed⌕ Search

Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 181 records · Page 10Linked to original sources
PubMed1993

Mediator release from mast cells by nerve growth factor. Neurotrophin specificity and receptor mediation.

Nerve growth factor causes mediator release from rat peritoneal mass cells in the presence of lysophosphatidylserine. We have investigated the neurotrophin and receptor specificity involved in this response. Nerve growth factor produced a dose-dependent release of [14C]serotonin in the presence of lysophosphatidylserine with an EC50 of approximately 1 nM. Incubation with brain-derived neurotrophic factor and neurotrophin-3 did not produce a response. Northern blot analysis with probes for low affinity nerve growth factor receptor (p75), trkA, trkB, and trkC demonstrated a detectable signal for trkA only. Western blots of trkA immunoprecipitates from mast cell culture lysates, probed with anti-phosphotyrosine antibodies, demonstrated expression of functional TrkA protein. To determine whether p75, trkB, or trkC mRNA was present in amounts below the limit of detection for Northern analysis, a sensitive reverse transcriptase polymerase chain reaction protocol was used; again rat peritoneal mast cells demonstrated only trkA. The predominant form of trkA message expressed in rat peritoneal mast cells was smaller than the neuronal form. An 18-nucleotide exon (coding for 6 amino acids in the extracellular domain) in the neuronal message was not found in the predominant mast cell trkA message. PC12 cells, a rat pheochromocytoma cell line, and dissociated rat sympathetic neurons showed both trkA and p75, but not trkB or trkC. Anterior pituitary expressed both trkB and trkC, but not trkA. To confirm the lack of expression of p75 on mast cells, 125I-nerve growth factor was chemically cross-linked to mast cells or PC12 cells and then immunoprecipitated with a monoclonal antibody specific for p75, 192-IgG; no p75 was detected. Thus, mediator release from rat peritoneal mast cells by nerve growth factor was specific and not a general property of neurotrophins, and the response was modulated through the trkA proto-oncogene. To our knowledge, this is the first description of a bone marrow-derived cell type that expresses trkA at both the mRNA and protein levels. These data provide further evidence that p75 is not necessary for nerve growth factor signal transduction.

Animals↗
PubMed1993

Neurotrophic factor deprivation-induced death.

Deprivation of sympathetic neurons of their physiological neurotrophic factor, nerve growth factor (NGF), leads to degeneration of soma and neurites, followed by loss of viability. The progression of degeneration and death are dependent upon macromolecular synthesis indicating an active participation of neuronal metabolism. Loss of viability begins only after a considerable delay after onset of NGF deprivation suggesting the presence of a sequence of degenerative events that triggers death. Such a sequence of degenerative events predicts that the activity of neuroprotective agents functioning by different mechanisms will be restricted to particular windows in time. The time-course of commitment to die as measured by the ability of NGF-deprived neurons to respond to NGF with long-term survival precedes the time-course of loss of viability by only a few hours, demonstrating that NGF displays neuroprotective properties for most of the time between onset of deprivation and death. Furthermore, NGF repairs and reverses the degenerative changes caused by prolonged periods of NGF deprivation. Because of these two aspects of NGF action, NGF demonstrates superior properties as a neuroprotective agents. NGF deprivation initiates DNA fragmentation of the neuronal genome into oligonucleosomal fragments in close temporal association with the onset of commitment to die. This is consistent with the idea that DNA fragmentation may be instrumental in causing the commitment to die. Thus, DNA fragmentation may serve as a marker of the physiologically most relevant critical step occurring during degeneration and may indicate the end of the period during which trophic factors are useful as neuroprotective agents. These results may be transferable to neurodegenerative diseases or sequelae of neuronal injury because of similarities in the phenomenology of degeneration and death.

Animals↗
PubMed1993

Transfection of human mesothelial cells mediated by different asbestos fiber types.

Several different asbestos fiber types mediate transfection of human mesothelial cells by exogenous DNA. We have employed the human MeT-5A mesothelial cell line, which allows the use of DNA replication as an assay for entry of DNA when plasmids bearing the SV40 origin of replication are used for transfection. We find that Canadian chrysotile, Calidria chrysotile, amosite, and crocidolite are each capable of introducing plasmid pSVod DNA into MeT-5A cells followed by subsequent replication of a fraction of the plasmid DNA. A significant fraction of the input plasmid DNA associated with the cells in the presence of asbestos is fragmented, and this fragmentation is particularly evident with crocidolite. Each of the fiber types is highly cytotoxic for the MeT-5A cells, and these cells actively accumulate the added fibers from the surrounding environment as visualized by phase-contrast microscopy. MeT-5A cells were transfected at higher efficiency with calcium phosphate than were several other primate cell lines. Calcium phosphate, however, did not induce fragmentation of the input plasmid DNA. Compared with several different mineral agents, including glass fibers, kaolin, and talc, Calidria chrysotile fibers were most effective at mediating transfection of the MeT-5A cells. Results provide a mechanism by which transfection can contribute to mutagenicity of asbestos fibers and indicate that this mechanism can operate in human mesothelial cells.

Asbestos↗
PubMed1993

NGF deprivation and neuronal degeneration trigger altered beta-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro.

In order to study the expression of beta-amyloid precursor protein (APP) isoforms during neuronal degeneration we have used the rat superior cervical ganglia (SCG) as an experimental model. In the neonate these sympathetic ganglia are nerve growth factor (NGF) dependent and in vivo administration of anti-NGF antiserum results in exaggerated neuronal degeneration. Analysis of APP mRNA transcripts in the SCG, following NGF deprivation, revealed a coincident decrease in APP695 and augmentation of APP751/770. These changes were specific to the SCG and were not seen in sensory ganglia. Subsequent in vitro studies, using primary dissociated cultures of sympathetic or cortical neurones, confirmed these changes in APP gene expression during neuronal degeneration. These observations may have important implications for the generation of beta-amyloid in Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1993

Cell death genes in invertebrates and (maybe) vertebrates.

That naturally occurring cell death in the nervous and other systems is an active and physiologically appropriate process has received much attention recently and has gained a significant degree of acceptance. The identification of cell death genes in invertebrates, the characterization of gene products that function as cell death suppressors, and the demonstration that some proto-oncogenes elicit cell death, as well as proliferation, in certain cell types have heightened interest in the mechanism of programmed cell death. Yet, evidence for a genetic program for cell death in vertebrates remains circumstantial and, so far, vertebrate 'cell death' genes exist only in theory.

Animals↗
PubMed1993

Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor.

The time course of molecular events that accompany degeneration and death after nerve growth factor (NGF) deprivation and neuroprotection by NGF and other agents was examined in cultures of NGF-dependent neonatal rat sympathetic neurons and compared to death by apoptosis. Within 12 h after onset of NGF deprivation, glucose uptake, protein synthesis, and RNA synthesis fell precipitously followed by a moderate decrease of mitochondrial function. The molecular mechanisms underlying the NGF deprivation-induced decrease of protein synthesis and neuronal death were compared and found to be different, demonstrating that this decrease of protein synthesis is insufficient to cause death subsequently. After these early changes and during the onset of neuronal atrophy, inhibition of protein synthesis ceased to halt neuronal degeneration while readdition of NGF or a cAMP analogue remained neuroprotective for 6 h. This suggests a model in which a putative killer protein reaches lethal levels several hours before the neurons cease to respond to readdition of NGF with survival and become committed to die. Preceding loss of viability by 5 h and concurrent with commitment to die, the neuronal DNA fragmented into oligonucleosomes. The temporal and pharmacological characteristics of DNA fragmentation is consistent with DNA fragmentation being part of the mechanism that commits the neuron to die. The antimitotic and neurotoxin cytosine arabinoside induced DNA fragmentation in the presence of NGF, supporting previous evidence that it mimicked NGF deprivation-induced death closely. Thus trophic factor deprivation-induced death occurs by apoptosis and is an example of programmed cell death.

Animals↗
PubMed1993

Fluconazole resistance in Candida glabrata.

We report a case of infection with Candida glabrata in which the organism became resistant to fluconazole and in which pre- and posttreatment isolates were available for comparison. The organism was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Fluconazole was a potent inhibitor of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) in lysates of cells from both susceptible and resistant cultures (50% inhibitory concentration, 0.2 microM), indicating that resistance was unrelated to changes in P-450DM. Instead, it appeared to arise from a permeability barrier to fluconazole, since resistant cells were unable to take up radiolabelled drug.

Aged↗
PubMed1993

Management of persistent vulvo vaginal candidosis due to azole-resistant Candida glabrata.

CASE REPORT--SUBJECTS--Three cases are described of long-standing vaginal candidosis due to Candida glabrata. These had failed to respond to local and systemic antifungals. In each case the infecting strain appeared resistant to a range of azole drugs in vitro. CLINICAL COURSE--Case one--This patient recovered following prolonged treatment with oral itraconazole in combination with oral and vaginal nystatin. Case two. Yeasts were eradicated from this patient following cyclical treatment with oral dydrogesterone; prolonged vaginal treatment with nystatin may have helped. Case three. This patient did not respond to a prolonged course of oral itraconazole in combination with vaginal and oral nystatin, oral medroxyprogesterone or intravaginal boric acid. Eradication of C glabrata was finally achieved by local application of 1% gentian violet. Shortly after eradication of the C glabrata infection, both Case two and Case three developed infections with other Candida species responsive to azole antifungals.

Administration, Oral↗
PubMed1993

Molecular mechanisms of developmental neuronal death.

Data derived from several experimental approaches demonstrate that naturally occurring neuronal death during development has many parallels with the physiologically appropriate death seen in nonneuronal cells. Physiologically appropriate death in different cell types may share some common mechanisms. These general notions must remain vague and tentative, because details of the mechanisms by which cells die in response to physiological positive or negative signals are poorly understood in any cell type. Current thinking focuses on the idea that cells possess a mechanism, which involves specific gene products, that are designed to kill the cell in response to appropriate physiological signals. Genetic studies of cell death in C. elegans and the demonstrations of increased expression of specific genes temporally associated with death in nonneuronal cells are consistent with this view. However, in the latter studies, there is no direct evidence that such temporally related genes are critical to the process of cell death or whether such gene expression may be related to somE other aspect of the response to the hormonal manipulations that produce the death of the cell under study. Therefore, the mechanism of death of any cell type is not understood, and whether neuronal death during development or after experimental manipulation results from the same mechanism is unknown. Several approaches are currently being pursued in a number of laboratories to address this general problem. These include pharmacological studies, such as described above, and studies aimed at analyzing biochemical and morphological changes associated with death. Attempts to find mRNAs or proteins whose increased expression is associated with neuronal death can be addressed by subtractive and differential hybridization strategies, by two-dimensional protein gel electrophoresis, and by examining genes whose increased expression is temporally correlated with cell death. Success in these various strategies will provide an understanding of neuronal death and relate to it cell death in other cell types. If future work provides direct evidence for a genetic program acting physiologically to produce death in the developing nervous system, an obvious question becomes the possible role that loss of transcriptional control of such a program plays in the adult in responses to mechanical or chemical trauma, neurodegenerative disease, or neuronal attrition associated with aging. Studies addressing the basic developmental process of trophic factor deprivation-induced death should provide molecular markers of and pharmacological approaches to these pathological processes in the adult.

Aging↗
PubMed1993

Eosinophilic mediastinitis, myositis, pleuritis, and pneumonia of cattle associated with migration of first-instar larvae of Hypoderma lineatum.

Migrating first-instar larvae of Hypoderma lineatum are a frequent cause of focal inflammatory lesions in connective tissues of the mediastinum, parietal and visceral pleura, peritoneum, lungs, diaphragm, and other loci. The lesions are characterized grossly by foci of yellowish or greenish gelatinous edema and microscopically by infiltration of the edematous tissue by a dense array of eosinophils. Lesions were recognized during a period of several weeks in late spring; the timing was attributable to events in the life cycle of the fly. The larvae, which were small (approximately 1 x 4.5 mm), transparent, and unobtrusive, were recovered from lesions in 12 of 20 cattle in which careful parasitologic examination was made.

Animals↗
PubMed1993

Structure-function relationships in rat brainstem subnucleus interpolaris. X. Mechanisms underlying enlarged spared whisker projections after infraorbital nerve injury at birth.

Prior studies indicate that the central projections of noninfraorbital vibrissae occupy greater than normal transverse areas in the rat trigeminal brainstem complex after infraorbital nerve section at birth. Here, we assessed the development of this phenomenon and possible underlying mechanisms. Cytochrome oxidase patches representing spared supraorbital (SO) or posteroorbital (PO) whiskers in the trigeminal subnucleus interpolaris (SpVi) were not reliably larger than those on the control side 24 hr after the infraorbital lesion. By 72 hr, SO and PO patches were 91% and 28% larger than those on the control side. Reliable increases were also observed on postnatal day 5 (PND5), PND7, and PND10 for the SO (59%, 65%, 66%) and PO (23%, 44%, 51%) patches. To test the hypothesis that central reorganization reflects the maintenance of peripheral supernumerary axons, myelinated and unmyelinated axons in SO vibrissa follicles were counted at PND0, PND7, PND17, and PND60. A corollary hypothesis, that peripheral regeneration errors result in both SO and surviving infraorbital axons, contributing to central SO patches, was tested with retrograde double-labeling methods. Both hypotheses were rejected. Thus, enlargement of SO patches is not due to either the maintenance of an immature peripheral innervation pattern, or regeneration of infraorbital axons into SO follicles. To determine if the enlargement of SO and PO patches produced by infraorbital nerve section is due to an activity-dependent competitive disadvantage imposed upon infraorbital afferents, TTX or bupivicaine was applied to the intact infraorbital nerve over the first 5-9 postnatal days. Brainstem maps developed normally and SO and PO patch areas were unaffected. Thus, impulse activity-based mechanisms do not appear to contribute to injury-induced patch enlargement. To test the hypothesis that patch enlargement is due to central collateral reorganization, intra-axonal recording and staining methods were applied to control and spared-whisker primary afferents in adult rats. Total bouton or collateral numbers did not differ in SpVi; however, arbor areas were reliably larger in experimental (14,879 +/- 350 microns 2) versus control (5527 +/- 1811 microns 2) fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1992

Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons.

Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited > or = 80%. When protein synthesis was inhibited within 22 +/- 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 +/- 4 h. Analogously, the commitment point for RNA synthesis was 26 +/- 4 h and that for NGF rescue, 24 +/- 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGF beta-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death.

Adenylate Kinase↗
PubMed1992

A "calcium set-point hypothesis" of neuronal dependence on neurotrophic factor.

In this commentary, we discuss evidence suggesting that cytoplasmic free calcium concentration determines neurotrophic factor dependence. Developing sympathetic and neural crest-derived sensory neurons require nerve growth factor (NGF) for survival both in vivo and in vitro. Chronic depolarization of these cells in culture causes a modest sustained elevation of cytoplasmic calcium concentration and promotes their survival in the absence of NGF. The amount of calcium increase caused by depolarization is closely correlated with the ability of the cells to survive in NGF-free medium. At an optimal calcium concentration, that we refer to as a "set point," survival is equivalent to that of cells grown in the presence of NGF.

Animals↗
PubMed1992

Activation of second-messenger pathways reactivates latent herpes simplex virus in neuronal cultures.

Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of cAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a cAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways.

Cell Line↗
PubMed1992

Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium.

Chronic depolarization greatly increases the survival of many types of neurons in culture. In at least some cases this enhancement of survival consists of the suppression of programmed cell death, a type of death occurring in developing neurons deprived of sufficient neurotrophic factor support. Available evidence suggests that the effect of depolarization on survival is mediated by a sustained rise of cytoplasmic free Ca2+, apparently caused by influx of Ca2+ through voltage-gated channels. This review discusses what is known about the mechanism by which prolonged depolarization and increased intracellular Ca2+ promote survival.

Animals↗