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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 271 records · Page 15Linked to original sources
PubMed1987

Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury.

The reaction of dorsal root ganglia (DRG) neurons to axotomy and its alteration by locally supplied nerve growth factor (NGF) were examined in adult rats. Surgically implanted silicone chambers attached to the severed tip of the sciatic nerve acted as reservoirs capable of providing prolonged access of NGF to the site of injury. The time course of NGF activity within the chambers was determined by using the standard NGF chick DRG bioassay. The fluid from chambers filled with the NGF-saline solution maintained NGF activity for periods up to 6 weeks after implantation. By 9 weeks, however, the fluid from most chambers failed to show any NGF activity in the bioassay. Experiments were designed to compare the response in adult rats to injury of DRG neurons receiving chambers filled with either NGF-saline or with only saline. The total neuronal counts in the lumbar fourth and fifth DRG at 3 weeks and 6 weeks after sciatic nerve section showed 22% and 16% cell death, respectively, in those injured neurons receiving saline-filled chamber implants. The animals that received chamber implants which contained an NGF-saline solution showed no cell death in the ipsilateral DRG at either 3 or 6 weeks after injury. Morphometric analysis of injured DRG neurons showed evidence of atrophy in the injured neurons which did not receive NGF. The degree of atrophy among all cell sizes was significantly decreased in those injured neurons receiving NGF. At 3 weeks after section the mean volume of injured neurons not treated with NGF was decreased by 28% as compared with only a 13% decrease in neurons treated with NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats.

The developmental expression of nerve growth factor (NGF) receptor was quantitated in either homogenates or plasma membrane-enriched preparations from whole rat embryos or from isolated tissues. The assay involved crosslinking 125I-NGF to receptors followed by immunoprecipitation with a monoclonal antibody to rat NGF receptor. In some cases, the pellet was resuspended and subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiographic analysis. The NGF receptor was found in whole embryo homogenates as early as embryonic Day 10 (E10) (earliest age examined). The NGF receptor content in whole embryos per milligram protein increased about 3-fold from E11 to E18 and decreased slightly at E20. SDS-PAGE autoradiography showed that the molecular weights of 125I-NGF-bound receptors did not vary with age. The NGF receptor content in sciatic nerve homogenates decreased 23-fold from newborn to adulthood. The change of NGF receptor level in hindleg muscle had a profile similar to that seen in sciatic nerve. The NGF receptor content in superior cervical ganglion (SCG) or dorsal root ganglion (DRG) homogenate preparations was expressed in two ways. On a per milligram protein basis, in SCG, the receptor density was decreased slightly from E20 to adulthood; in DRG, it was relatively constant from E15 through postnatal Day 0 (PND-0) and then dropped 6.7-fold in adults. On a per ganglion basis, in SCG, it increased 4.4-fold from E20 to adult; in DRG, it increased 9-fold from E15 to PND-0 and then stayed constant through adulthood. In brain membrane preparations, the NGF receptor level decreased 11-fold from E15 to adulthood. In spinal cord membrane preparations, it decreased 7-fold from E18 to adulthood. Levels of receptor in cord were always greater than in brain. These data suggest that alterations in the NGF receptor density may have a role in changes in tissue responsiveness to NGF during development.

Age Factors↗
PubMed1987

Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study.

The distribution of nerve growth factor receptors in the lumbar spinal cord of the rat was studied with autoradiographic and immunohistochemical techniques: [125I]nerve growth factor and specific monoclonal antibody (Mab 192) against nerve growth factor receptor were used to localize nerve growth factor binding sites. The distributions of nerve growth factor binding sites with highest density within the superficial layers (laminae I and II) of the dorsal gray matter were virtually identical as demonstrated by these two ligands; this suggests that Mab 192 can be used as a specific probe to identify nerve growth factor receptors in rat nervous system. Nerve growth factor receptor binding sites, as demonstrated by autoradiography, were also found in longitudinal bundles of fibers running dorsolaterally in the lateral funiculus. However, no immunoreactivity was detected in these areas by immunohistochemistry. No specific binding was found in the dorsal horn when [125I]nerve growth factor was co-injected with unlabeled nerve growth factor or after incubation with nonspecific monoclonal antibody. Dorsal root section produced a complete loss of nerve growth factor-specific labeling pattern throughout laminae I-II of the spinal cord. This suggests that nerve growth factor receptors are localized on the nerve terminals of primary afferent fibers which synapse in the region of the spinal cord. The presence of nerve growth factor binding sites in the dorsal horn of the spinal cord is consistent with the possibility that nerve growth factor, or a nerve growth factor-like substance, derived from the central nervous system, may have a role in trophic support of dorsal root ganglion neurons.

Afferent Pathways↗
PubMed1987

Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system.

NGF acts on responsive neurons by binding to specific NGF receptors on axonal termini, after which a critical biochemical signal is retrogradely transported to the cell body. The identity of the signal(s) is unknown; candidates include NGF itself or some other "second messenger." A possible second messenger is the NGF receptor. As a first step in assessing the possible role of NGF receptor in the generation of the NGF-dependent signal, and in understanding the economy of NGF receptor synthesis and utilization, we determined whether the NGF receptor is retrogradely transported. Using immunohistochemical staining with a monoclonal antibody (192-IgG) against rat NGF receptor, we looked for accumulation of NGF receptor molecules distal (retrograde transport), as well as proximal (anterograde transport), to sites of axonal ligation or transection. By 10-12 hr in both the ligated sciatic nerve and the lesioned fimbria-fornix, accumulated NGF receptor was detected proximal and distal to the ligation/lesion site. The transported receptor presumably was located in sympathetic and sensory neurons in the sciatic nerve and in forebrain cholinergic neurons projecting from the medial septum to the hippocampus. In both anatomical sites, accumulation of NGF receptor on the proximal (anterograde) side occurred in streams of fine axonal processes, whereas staining on the distal (retrograde) side occurred in varicose or granular configurations. These results raise the possibility that the NGF receptor has a role in the mechanism of NGF beyond the initial binding event at the plasma membrane of the axonal terminus.

Animals↗
PubMed1987

MHC-specific cytotoxic T lymphocyte killing of dissociated sympathetic neuronal cultures.

Experiments were conducted to determine whether neurons in culture can serve as targets for immunologic attack mediated by major histocompatibility complex (MHC)-specific cytotoxic T lymphocytes (CTLs) which recognize Class I antigens. Allogeneic C3H/He primary neuronal cultures were quickly destroyed after CTL addition, while syngeneic C57BL/6J neurons were not lysed. Alterations in the distribution of chromatin were the first ultrastructural changes that occurred, followed by loss of nuclear morphology, cytosolic changes, and eventually fragmentation of both the nucleus and cytosol. With Campenot chambers, it was possible to separate the membrane and nuclear lesions. CTLs exposed to neurites, but separated from the cell body by the chamber barrier, caused degeneration of neurites but did not cause lysis and cell death. Neuronal lysis mediated by antibody and complement appeared to be distinct from CTL-mediated lysis. These experiments demonstrate that neurons in culture are targets for MHC-specific CTLs, and therefore probably express functional levels of Class I antigens. The signal for killing by CTLs is not retrogradely transported from the neurite to the cell body, and morphologic events following CTL-neuron interaction resemble those that occur in dividing tumor target cell populations.

Animals↗
PubMed1986

Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3.

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.

Adrenal Gland Neoplasms↗
PubMed1986

Species and structural specificity of the lipopigment accumulation and neuronal destruction induced by N-(2-guanidinoethyl)-4-methyl-1,2,5,6-tetrahydropyridine (guanacline).

Guanacline, a guanidinium adrenergic neuron blocking agent similar to guanethidine, was studied clinically and experimentally during the late 1960s. Like guanethidine, it has been reported to produce sympathetic neuronal destruction in rats. Unlike guanethidine, it has been reported to produce irreversible sympathetic deficits in man and to produce fluorescent lipopigment in rat sympathetic neurons. Guanacline and its derivative in which the double bond of the tetrahydropyridine ring is reduced (saturated analog of guanacline, SAG) were prepared. Several species were treated chronically with varying doses of guanethidine, guanacline or SAG; the superior cervical ganglia were examined light microscopically for neuronal destruction and for osmiophilic fluorescent lipopigment accumulation. All 3 drugs produced rapid neuronal destruction in rats accompanied by massive small-cell infiltration. In striking contrast, treatment for many weeks with doses up to 100 mg/kg/day produced no small-cell infiltration or apparent neuronal destruction in mice or guinea pigs. The neuronal destruction produced by guanacline and SAG in the rat, like that caused by guanethidine, was prevented by immunosuppression or gamma-irradiation, indicating that all 3 agents produce neuronal destruction in rats by an immune-mediated mechanism. Thus, the ability of the drug to produce sympathectomy is species specific but not drug specific. The opposite was found with respect to fluorescent lipopigment accumulation. Guanacline, but not guanethidine or SAG, produced fluorescent lipopigment in all species examined. Therefore, the double bond of the tetrahydropyridine ring plays a critical role in the production of the fluorescent lipopigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Characterization of choline acetyltransferase-sustaining and survival-promoting activities for parasympathetic neurons in pig lung.

A total of 49 tissues from six animal species were screened for substances that would sustain choline acetyltransferase (ChAT) in parasympathetic ciliary neurons in culture. Among these, lungs from several species, particularly pig, contained relatively high concentrations of ChAT-sustaining activity. The bioassay used was one in which embryonic chicken ciliary ganglia were cultured as explants for 3 days with KCl and lung extract. KCl, in the presence of lung extract, sustained high concentrations of choline acetyltransferase activity, but did not maintain enzymatic activity in the absence of lung extract. These effects were dependent on the embryonic age of the ciliary ganglia. Lung extract and KCl would not maintain choline acetyltransferase activity in parasympathetic neurons taken from chickens older than embryonic day 12. In addition to ChAT-sustaining activity, pig lung also contains survival-promoting activity for parasympathetic neurons but not for other neuronal types, including sympathetic or sensory neurons of neural crest or placodal origin. This is a more specific target spectrum than previously reported ciliary survival-promoting factors that have been purified or partially purified and indicates that survival-promoting activities in pig lung are different than those previously observed. Because of the large quantitites that can be obtained, we suggest that pig lung is a preparatively feasible source for possibly novel ciliary neuron ChAT-sustaining and survival-promoting activities for parasympathetic neurons.

Animals↗
PubMed1986

Comparative dynamics of retrograde transport of nerve growth factor and horseradish peroxidase in rat lumbar dorsal root ganglia.

The dynamics of the retrograde transport of [125I] nerve growth factor (NGF) and horseradish peroxidase (HRP) in dorsal root ganglion (DRG) neurons were studied in rats. After injection of [125I]NGF or HRP into crushed sciatic nerve, labelling was examined in spinal nerves, dorsal root ganglia, dorsal roots and spinal cord. Retrograde transport of either [125I]NGF or HRP was first observed in DRG neurons 6 h after injection: The maximal rate of transport (7 mm h-1) was similar for both proteins. Significant differences in the sizes of DRG neurons labelled by [125I]NGF were observed and were dependent upon survival time. No such difference was seen in HRP-injected animals. At 6 h after injection, 60% of all the HRP-labelled cells had a diameter of more than 25 micron, whereas 90% of all the [125I]NGF-labelled neurons had a diameter of less than 25 micron. With increasing survival times there was a gradual shift in the size of [125I]NGF-labelled neurons towards larger diameters. Thus, 24 h after the [125I]NGF injection, 83% of the labelled cells had a diameter greater than 25 micron. The data suggest that small diameter neurons retrogradely transport and turnover NGF faster than larger diameter neurons. There was a preferential accumulation of silver grains in small DRG neurons (mean diameter 25 micron) at early survival times (4 and 8 h); at the later survival time (24 h) the reverse was observed, i.e. larger neurons (mean diameter 42 micron) were labelled. In contrast, the mean diameter of HRP-labelled neurons remained constant (30 micron) at all times after injection. The total number of neurons ultimately labelled (approximately 80-85%) appeared to be the same with both tracers. In addition, the lack of transganglionic transport of NGF into the spinal cord and the short time span of the observable accumulated radioactivity in DRG neurons suggest the rate of degradation of transported NGF seems to be faster than HRP. As a practical matter, these data indicate that observing cells within DRG which accumulate retrogradely transported [125I]NGF at any one time gives an inaccurate picture of the size properties of cells capable of transporting the ligand.

Animals↗
PubMed1986

The developmental toxicity of xylene and xylene isomers in the Hydra assay.

Two laboratories tested multiple forms of xylene for their developmental toxicity hazard potential (A/D ratio) by means of the hydra assay. The three isomers, as well as a solution of mixed xylenes, all interfered with development (D) at or near to concentrations that also were toxic to adult (A) hydra. The A/D ratios ranged from 1 to 2 in hydra as they had in conventional tests made in pregnant laboratory animals. Each testing laboratory concluded that xylenes were not primary developmental hazards but coaffective agents capable of disrupting development only at or near to concentrations also toxic to adults. In each instance every xylene tested interfered with the same stage or developmental sequence and in a concentration-related manner. The hydra assay may be useful for establishing priorities to test agents in a more elaborate system, but substances less soluble than xylene may exceed the test's applicability.

Animals↗
PubMed1986

Induction of nerve growth factor receptor in Schwann cells after axotomy.

We have discovered that axotomy of sciatic nerve induces Schwann cells distal to the lesion to express de novo, or at greatly increased levels, receptors for nerve growth factor (NGF). Surgical transection of sciatic nerve was performed on adult Sprague-Dawley rats, and, at various times after the operation, the following tissues were dissected for quantitation of NGF receptor: L4 and L5 dorsal root ganglia, sciatic nerve proximal to the transection, sciatic nerve distal to the transection, tibialis anterior muscle, and skin of the dorsum of the foot. The NGF receptor content of these samples was determined by labeling receptor molecules with radioiodinated NGF (125I-NGF) and then specifically immunoprecipitating the 125I-NGF-receptor complexes with 192-IgG, a monoclonal antibody directed against the rat NGF receptor. We observed a time-dependent increase in the amount of radioligand-labeled NGF receptor proteins found in the distal segment of transected sciatic nerve; by 7 days the density of receptor (crosslinked 125I-NGF molecules per mg of homogenate protein) had increased at least 50-fold. The number of receptor molecules in tibialis anterior muscle and dorsal foot skin, two structures denervated by the transection, also increased, with time courses parallel to that of distal sciatic nerve. There was little increase in the density of NGF receptors in the sciatic nerve proximal to the lesion and in the dorsal root ganglia. Immunohistochemical examination of the distal portion of transected sciatic nerve and of the muscle, with 192-IgG as the primary ligand, revealed prominent and exclusive staining of apparently all Schwann cells of the endoneurium, indicating that these peripheral neuroglial cells were expressing NGF receptors. These results show that axonal damage can induce the expression of NGF receptors in the population of sheath cells thought to promote neuronal regeneration. This dramatic increase in NGF receptors may be a mechanism to facilitate the regeneration of NGF-responsive neurons.

Animals↗
PubMed1986

Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells.

Monkey COS-7 cells were transformed with BLUR8 DNA, a pBR322 plasmid containing a human Alu-family sequence at the BamHI site. Within 24 hr of transformation 2-5% of the BLUR8 molecules recovered resisted cleavage by Dpn I, indicating they had replicated. Electron microscopy revealed appropriately sized circular molecules with replication bubbles whose centers were mapped to the Alu insert. A 16-base-pair deletion within the Alu sequence prevented replication. The results indicate that certain Alu sequences can serve as origins of replication in COS-7 cells.

Animals↗
PubMed1986

Nerve growth factor receptor molecules in rat brain.

We have developed a method to immunoprecipitate rat nerve growth factor (NGF) receptor proteins and have applied the method to detect NGF receptor molecules in the rat brain. Crosslinking 125I-labeled NGF to either PC12 cells or cultured rat sympathetic neurons yielded two radiolabeled molecules (90 kDa and 220 kDa) that were immunoprecipitated by monoclonal antibody 192-IgG. Further, 192-IgG precipitated two radiolabeled proteins, with the expected sizes (80 kDa and 210 kDa) of noncrosslinked NGF receptor components, from among numerous surface-iodinated PC12 cell proteins. These results demonstrate the specific immunoprecipitation of NGF receptor molecules by 192-IgG. We applied the 125I-NGF crosslinking and 192-IgG-mediated immunoprecipitation procedures to plasma membrane preparations of the following areas of rat brain: medial septum, cerebellum, brainstem, hippocampus, cerebral cortex, thalamus, and olfactory bulb. NGF receptor molecules of the same molecular masses as the peripheral receptor components were consistently detected in all of these regions and in preparations from whole brains. Removal of the peripheral sympathetic innervation of the brain did not eliminate these NGF receptor proteins, indicating that the receptor is endogenous to central nervous system tissues. We also observed retrograde transport of 125I-labeled 192-IgG from the parietal cortex to the nucleus basalis and from the hippocampus to the nucleus of the diagonal band of Broca and the medial septal nucleus. These findings demonstrate the presence in brain of NGF receptor molecules indistinguishable from those of the peripheral nervous system.

Animals↗