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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 217 records · Page 12Linked to original sources
PubMed1990

Structure and expression of cytochrome f in an Oenothera plastome mutant.

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Base Sequence↗
PubMed1990

Nerve growth factor receptor in neural lobe of rat pituitary gland: immunohistochemical localization, biochemical characterization and regulation.

Nerve growth factor-receptor immunoreactivity was detected in the neural lobe of the pituitary gland in developing and adult rats of both sexes. The presence of nerve growth factor receptor in the neural lobe was further verified by a quantitative 125I-nerve growth factor/crosslink/immunoprecipitation assay and subsequent visualization by SDS-PAGE autoradiography. Nerve growth factor-receptor immunoreactivity was detected in the neural lobe of postnatal 5-day-old rats, had increased by 2 months and was much higher in 1-year-old rats. In 2-month-old rats, no immunoreactivity was observed in anterior or intermediate lobes. Pituitary stalk transection in young adult rats greatly increased the expression of nerve growth factor-receptor immunoreactivity in the neural lobe, although the staining pattern remained the same. This increase began 3 days after surgery, and reached peak levels at approximately 15 days. Other physiological or non-physiological changes did not alter the nerve growth factor-receptor immunoreactivity in the neural lobe; these changes included dehydration, pregnancy and lactation, castration of male rats, bilateral superior cervical ganglionectomy and intraventricular injection of colchicine. Intravenously injected 125I-nerve growth factor was specifically accumulated in both normal and denervated neural lobe. Nerve growth factor-receptor immunohistochemical electron microscopy showed that the receptor-positive cells are fusiform and found both inside and outside the basal lamina that delimits the neural lobe parenchyma. Based upon the anatomical localization, morphology and response to axotomy, we identify, at least the perivascular component, as microglia. These data suggest a role for nerve growth factor and/or nerve growth factor receptor in microglial function.

Animals↗
PubMed1990

Computer programs for the analysis of immunohistochemically labeled spinal cord sections.

A microcomputer based image processing system has been developed to analyze the density of immunohistochemical labeling within neural tissue. The system permits the tracing of anatomical regions seen on a digitized image and the determination of the area and the mean gray level of all pixels lying within each defined region. Corrections are made for uneven screen illumination and for nonlinearity of the video camera's response to gradations in optical density. A standardization procedure ensures that the same level of incident illumination is used during each experiment. An example of an application of this system is described in which unilateral changes in immunohistochemical staining density were determined within the rodent spinal cord following partial spinal injury. This system provides a sensitive and objective means of analyzing such changes.

Animals↗
PubMed1990

The in vitro activation of cyclophosphamide in the Hydra Developmental Toxicology Assay.

The proteratogen cyclophosphamide (CP) was tested in the Hydra Assay in the presence and absence of an in vitro metabolic activation package (MAP) consisting of rat hepatic microsomes (0.06 nmol P450/ml), 500 microM NADPH, and 25 microM MgCl. This metabolic system was developed through a series of interrelated biochemical and biological assays to provide maximum cytochrome P450 mixed-function monooxygenase (MFO) metabolic capacity while controlling the inherent toxicity of the hepatic preparation and the attendant cofactors. Bioactivation of CP was confirmed under standard hydra assay conditions of pH 7.0 and 20 degrees C and compared with activation at 37 degrees C. Estimation of total metabolic capacity and verification of activation were made through the appearance of alkylating metabolites both in the absence and in the presence of hydra. Chemical exposure was maintained throughout the 92 +/- 2 hr assay with periodic renewal of media (and additives) at 4, 20, 28, 42, and 66 hr of incubation. Inclusion of bioactivation increased the toxicity of CP by two orders of magnitude. The minimal affective concentration in the adult and developmental portions of the assay was decreased from 4000 to 20 micrograms CP/ml and from 1000 to 4 micrograms CP/ml, respectively. By limiting the inherent toxicity of the MFO package, it was possible to avoid pulse-type exposures and ensure that all ontogenic stages were exposed to active metabolites. The addition of metabolic activation capacity to an in vitro assay, while not essential, markedly enhances its utility and breadth of application in developmental toxicity safety evaluations.

Animals↗
PubMed1990

Prediction of the developmental toxicity hazard potential of halogenated drinking water disinfection by-products tested by the in vitro hydra assay.

A series of seven randomly selected potential halogenated water disinfection by-products were evaluated in vitro by the hydra assay to determine their developmental toxicity hazard potential. For six of the chemicals tested by this assay (dibromoacetonitrile; trichloroacetonitrile; 2-chlorophenol; 2,4,6-trichlorophenol; trichloroacetic acid; dichloroacetone) it was predicted that they would be generally equally toxic to both adult and embryonic mammals when studied by means of standard developmental toxicity "teratology" tests. However, the potential water disinfection by-product chloroacetic acid (CA) was determined to be over eight times more toxic to the "embryonic" developmental portion of the assay than it was to the adults. Because of this potential selectivity, CA is a high-priority item for developmental toxicity tests in pregnant mammals to confirm or refute its apparent unique developmental hazard potential and/or to establish a NOAEL by the route of most likely human exposure.

Animals↗
PubMed1990

Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization.

Fibroblast growth factor receptor (FGF-R) mRNA expression was examined in the adult rat CNS. Northern blot analysis showed a distinct 4.3 kb transcript in various CNS regions. In situ hybridization revealed widely distributed, but specific, populations of cells that express FGF-R mRNA. The most intense hybridization signals were observed in the hippocampus and in the pontine cholinergic neurons. The limbic system and brainstem nuclei, including motor nuclei, showed robust labeling. Cerebellar granule cells and spinal cord neurons were positive for FGF-R mRNA. The distribution of FGF-R mRNA differed significantly from that of NGF receptor mRNA; particularly, no hybridization signal was detected in basal forebrain cholinergic neurons. These results strongly suggest that FGF or FGF-like molecules may exert effects on specific neuronal populations in the mature CNS.

Amino Acid Sequence↗
PubMed1990

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant.

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.

Journal Article↗
PubMed1990

Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases.

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

Amino Acid Sequence↗
PubMed1990

Interferon suppresses sympathetic neuronal cell death caused by nerve growth factor deprivation.

Cultured rat sympathetic neurons die within 48 h after being deprived of nerve growth factor. Addition of interferons (IFN-alpha/beta or IFN-gamma) prevented the cell death in a dose-dependent manner. Upon longer periods of nerve growth factor deprivation, IFNs failed to maintain survival. Thus, IFNs retarded neuronal death, but did not prevent it. Ligand binding, autoradiography, and cross-linking experiments demonstrated the presence of specific IFN-gamma receptors on sympathetic neurons similar to those seen on other cell types. The possible relationships of the death-suppressing actions of IFNs are compared to the mechanisms of the antiviral or antiproliferative actions of IFNs.

Adenylate Kinase↗
PubMed1990

An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells.

Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.

Cell Division↗
PubMed1990

Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction.

Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis. In this paper we report that AraC kills postmitotic rat sympathetic neurons in a fashion similar to the neuronal death that follows nerve growth factor (NGF) deprivation. Postmitotic rat sympathetic neurons were cultured for 1 week in the presence of NGF and then treated with AraC, still in the presence of NGF. AraC killed neurons after 4 d with an EC50 of 50 microns. The morphological and temporal characteristics of neuronal death that began around 3 d after addition of AraC were indistinguishable from those observed beginning 24 hr after NGF deprivation. Death caused by AraC was prevented by the same agents that prevent the death of NGF-deprived neurons, which included inhibitors of RNA and protein synthesis, a cAMP analog, and depolarizing concentrations of KCl. In contrast, neuronal death caused by ultraviolet irradiation, ricin toxin, and a variety of other toxic insults did not share these morphological, biochemical, or temporal characteristics. Other antimitotic drugs, including adenine arabinoside, thymine arabinoside, fluorodeoxyuridine, hydroxyurea, and aphidicolin, did not kill neurons. AraC caused neurons to behave as if deprived of NGF by interfering with deoxycytidine (dC) metabolism distinct from DNA biosynthesis. dC entirely prevented the neurotoxicity of AraC, even when present at a concentration 1000-fold less than that of AraC. Other deoxynucleosides, and cytidine, did not prevent AraC neurotoxicity. dC could not, however, substitute for NGF and thus is unlikely to be a direct mediator of NGF action. It is hypothesized that dC may participate in a pathway, distinct from DNA synthesis, that is necessary for neurons to respond to exogenous trophic factors.

Animals↗
PubMed1990

Basic fibroblast growth factor: receptor-mediated internalization, metabolism, and anterograde axonal transport in retinal ganglion cells.

Basic fibroblast growth factor (bFGF) was radiolabeled and used in axonal transport studies to determine whether certain neuronal populations express functional receptors for bFGF. Unlike 125I-NGF, 125I-bFGF was not retrogradely transported in the adult rat sciatic nerve or from iris to trigeminal ganglion or superior cervical ganglion. However, after intraocular injection of 125I-bFGF into the posterior chamber of the eye of adult rats, radioactivity was detected within the retinal ganglion cell projections. This radioactivity was localized to the ipsilateral optic nerve and in the contralateral lateral geniculate body and the contralateral superior colliculus by using autoradiographic techniques. Direct measurement of the radioactivity in dissected brain regions was used to study the process of 125I-bFGF uptake and transport by retinal ganglion cells. The uptake and transport were specific for biologically active bFGF since neither denatured, biologically inactive 125I-bFGF nor 125I-NGF was taken up and transported. The uptake and transport of 125I-bFGF were saturable phenomena since they were blocked in the presence of excess, unlabeled bFGF. Wheat germ agglutinin, but not heparinase, blocked uptake and transport of 125I-bFGF, a finding that is consistent with the uptake being mediated by high-affinity bFGF receptors. Radioactivity from 125I-bFGF was transported in retinal ganglion cell axons in an anterograde direction at a maximum rate in excess of 1.7 mm/hr. No specific retrograde transport of bFGF to the retina was detected after 125I-bFGF was injected into the superior colliculus. The radioactivity from 125I-bFGF that accumulated in the superior colliculus was lost from this tissue with a half-life of about 22 hr. Autoradiography of proteins separated by SDS-PAGE demonstrated that 125I-bFGF was not substantially degraded in the retina after internalization within retinal ganglion cells. During anterograde transport, however, 125I-bFGF underwent limited proteolytic cleavage resulting in 3 prominent 125I-bFGF derivatives of molecular weights greater than 7000 Da. Although these were the major radioactive species recovered from the superior colliculus after intraocular injection, some intact 125I-bFGF was also detected within the innervated target. These results indicate that retinal ganglion cells express high-affinity receptors for bFGF, that these receptors mediate the internalization of bFGF, that internalized bFGF undergoes limited proteolytic cleavage, and that bFGF and its derivatives are anterogradely transported to the lateral geniculate body and the superior colliculus. These data raise the possibility that bFGF or its derivatives may act as an anterograde trophic factor in the visual system, a system that is known to undergo anterograde transneuronal cell death.

Animals↗
PubMed1990

Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro.

Previously, we reported that nerve growth factor (NGF) is required to maintain herpes simplex virus (HSV) latency in cultures of rat sympathetic neurons (Wilcox and Johnson, 1987, 1988). Here, we extend these results by showing that NGF was also required to maintain HSV latency in cultures of sensory neurons obtained from dorsal root ganglia of rats, monkeys, and humans. The interruption of the neuronal supply of NGF for 1 hr reactivated HSV, indicating that the latent virus was exquisitely sensitive to perturbations in the concentration or binding of NGF. A species-specific monoclonal antibody directed against the human NGF-receptor, which blocks NGF binding, reactivated latent HSV in human, but not rat, sensory neurons. In contrast, a monoclonal antibody against the rat NGF-receptor, which binds the receptor without blocking NGF action, did not produce reactivation. These results indicate that the effects of NGF on HSV latency are mediated via NGF binding to the NGF receptor. In addition, treatments that interfere with specific steps in the transduction of the NGF signal, including treatment with 6-hydroxydopamine and colchicine, reactivated latent HSV. Further, in neurons harboring latent virus, interruption of protein synthesis or RNA transcription for 1 hr resulted in viral reactivation, suggesting that a short-lived factor may be present in neurons which represses viral reactivation.

Animals↗
PubMed1990

Safety of Sanguinaria extract as used in commercial toothpaste and oral rinse products.

This report represents the findings of an Expert Panel on the safety of Sanguinaria extract used in Viadent oral rinse and toothpaste products and represents an independent review of the Sanguinaria extract toxicologic data base. It is based on reviews and discussions of the data base by all members of the Expert Panel on Sanguinaria extract. The Panel concluded that the data base on Sanguinaria extract is substantial and indicates that Sanguinaria extract is safe in its present use in Viadent products based on a large margin of safety between levels of human exposure and levels found to produce minimum effect or to be without adverse effect in animals. The panel further concluded that published literature suggesting an association between human exposure to Sanguinaria extract and potential reproductive, cardiovascular, or ocular toxicity, or carcinogenicity is largely anecdotal, unfounded, and not corroborated by or consistent with the substantial data base that was subjected to peer review.

Alkaloids↗
PubMed1990

Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse.

We have investigated the effects of prolonged systemic injections of nerve growth factor (NGF) and its antiserum on the survival and morphology of sympathetic ganglion cells in adult mice. Using intracellular injections of Lucifer yellow in lightly fixed superior cervical ganglia, we show that total dendritic lengths of ganglion cells are increased 29% after 2 weeks of NGF treatment. The increased dendritic length is characterized by increased branching within the dendritic arborization and not by the addition of new primary dendrites. In addition, cell soma cross-sectional area was increased 45%. Conversely, administration of NGF antiserum for 1 month decreased total dendritic length by 33%, decreased ganglion cell body size by 26%, and reduced the number of neurons in the ganglion by 25%. After 3 months of NGF antiserum treatment, the number of neurons in the ganglion was reduced a total of 41%. NGF antiserum treatment for 1 month in aged (22 months old) animals reduced ganglion cell body size by 21% and cell number by 22%, decreases that are comparable to those observed in young adult animals. Our results indicate that, even in maturity, sympathetic ganglion cells remain dependent on NGF for survival and maintenance of dendritic geometry, and this dependence continues into old age.

Animals↗
PubMed1989

Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain.

The expression of nerve growth factor (NGF) receptor in adult rat brain was studied by immunohistochemistry with a specific anti-rat NGF receptor monoclonal antibody, 192-IgG. Intense NGF receptor immunoreactivity (NGFRI) was found in structures known to be NGF responsive, including forebrain cholinergic neurons in medial septum, diagonal band of Broca, and basal nucleus of Meynert; central processes of neural-crest-derived sensory ganglion neurons and their innervated nucleus also contained such immunoreactivity. Distinct NGFRI staining was also found in many brain areas and cell types not known to be NGF responsive, including some hypothalamic regions, circumventricular organs, some areas related to the optic system, olfactory glomeruli, ependymal and subependymal cells in some locations, mesencephalic nucleus of the trigeminal nerve, cerebellar molecular layer, central linear nucleus, solitary tract and its nucleus, and inferior olive. The NGFRI in the circumventricular organs was further studied by in vivo labeling of 125I-ligands. Intravenously injected 125I-NGF, but not 125I-cytochrome c, was specifically accumulated in the area postrema. Biochemical study of the NGF receptor showed a major band of molecular weight of approximately 90 KDa in the area postrema, choroid plexus, median eminence, and medial septum with the relative content consistent with that seen by immunohistochemistry. No evidence of a truncated NGF receptor was observed. The results of this study suggest that NGF and its receptor have broader roles in adult mammalian brain than previously thought.

Animals↗
PubMed1989

Selective initiation of replication at origin sequences of the rDNA molecule of Physarum polycephalum using synchronous plasmodial extracts.

A cell-free system using synchronous plasmodial extracts initiates replication selectively on the 60 kb rDNA palindrome of Physarum polycephalum. Preferential labeling of rDNA fragments by nuclear extracts, in which elongation is limited, indicates that initiation occurs at two positions corresponding to in vivo origins of replication estimated by electron microscopy. Both nuclear and whole plasmodial extracts initiate selectively within a plasmid, pPHR21, containing one of these origins. In this plasmid bubbles expand bidirectionally and generate DpnI-resistant DNA. Extracts made at prophase or early S phase, times when the nucleolus is disorganized, are most active in pPHR21 replication. Mapping positions of replication bubbles locates the initiation point in a 3.2 kb BstEII fragment at the upstream border of a series of 31 bp repeats 2.4 kb from the initiation point for ribosomal gene transcription.

Base Sequence↗