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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 163 records · Page 9Linked to original sources
PubMed1995

Localization of PURA, the gene encoding the sequence-specific single-stranded-DNA-binding protein Pur alpha, to chromosome band 5q31.

Pur alpha (PurA) is a sequence-specific single-stranded-DNA-binding protein implicated in control of both DNA replication and transcription. We have localized the Pur alpha gene (PURA) to human chromosome band 5q31 by fluorescence in situ hybridization with a 16-kb genomic probe together with hybridization of a cDNA probe to blots of DNA from human-hamster cell lines containing individual human chromosomes. Sequences with homology to the PURA locus are also present at 6q14. The 5q31 locus is frequently deleted in myelogenous leukemia and other cancers.

Animals↗
PubMed1995

Substance abuse and violence: cause and consequence.

Substance abuse has been associated with violent behavior for many decades. While the relationship is the same today as it was in the past, the pervasiveness of the association, and the consequences, are more dramatic. There are two ways in which substance abuse is related to violence. First, violence can be and is perpetrated under the influence of substances, and second, violence related to substance abuse stems from the trade in drugs, which is all too often focused in poor and underserved communities. The elimination of the market for drugs, and thus the reduction in the demand for drugs, will bring about a reduction in substance abuse-related violence.

Adolescent↗
PubMed1995

Chronic depolarization prevents programmed death of sympathetic neurons in vitro but does not support growth: requirement for Ca2+ influx but not Trk activation.

Continuous exposure of many types of neurons in cell culture to elevated concentrations of K+ greatly enhances their survival. This effect has been reported to be mediated by a sustained rise of cytoplasmic free Ca2+ concentration caused by influx of Ca2+ through voltage-gated channels activated by K(+)-induced chronic depolarization. In this report we investigate the effects of elevated K+ on the programmed death that embryonic rat sympathetic neurons undergo in culture when deprived of NGF. Elevated K+ in the culture medium did not significantly prevent death of NGF-deprived cells until after the third day following plating of embryonic day 21 neurons. On the fifth day after plating, incrementally increasing K+ concentrations in the culture medium from 5 to 100 mM caused chronic depolarization of neurons and had a biphasic effect on survival of NGF-deprived cells. Enhanced survival was steeply related to membrane potential, increasing from no enhanced survival in cells held at potentials between -51 and -34 mV to 90-100% of control survival at about -21 mV. At potentials positive to -21 mV, survival decreased. Associated with the chronic depolarization was a sustained rise of steady-state free Ca2+ concentration that showed a biphasic relationship to membrane potential roughly similar to that exhibited by survival. Steady-state Ca2+ concentration increased with increasingly lower membrane potentials to a peak at about -23 mV (to approximately 240 nM from approximately 40 nM at about -51 mV) and then decreased at more positive potentials. The elevation of intracellular Ca2+ was largely blocked by dihydropyridine and phenylalkylamine Ca2+ channel antagonists and was potentiated by a dihydropyridine Ca2+ channel agonist. Neither the rise of Ca2+, or survival was affected by the Ca2+ channel antagonist, omega-conotoxin. Therefore, the Ca2+ elevation was probably caused by Ca2+ influx through L-type, but not N-type, channels. Antagonists of L channels blocked both survival and the sustained increase of steady-state free Ca2+ at similar concentrations, suggesting that the relevant factor determining survival of depolarized cells was Ca2+ influx rather than some other effect of depolarization. Surprisingly, however, there was no clear correlation between the sustained rise of Ca2+ and survival. Some membrane potentials that induced similar increases of Ca2+ concentration produced widely different levels of survival. While chronic depolarization promoted survival of neurons in the absence of NGF, cells supported in this manner showed little growth as measured by neurite extension, total cellular protein, and mean somal diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Conservation in human and mouse Pur alpha of a motif common to several proteins involved in initiation of DNA replication.

Pur alpha is a sequence-specific single-stranded DNA-binding protein with affinity for an element present in several eukaryotic origins of DNA replication (ori) and gene regulatory regions. We report here the cDNA sequence for mouse pur alpha and an extraordinary degree of conservation between human and mouse Pur alpha (hPurA and mPurA, respectively). There are only two single-amino-acid (aa) changes between hPurA (322 aa) and mPurA (321 aa). One PurA region of 22 aa, termed the 'psycho' motif, possesses significant homology to a counterpart in the SV40 large T-antigen, to several other transforming proteins of DNA tumor viruses, and to certain cellular proteins in yeast and human cells that may also be involved in the initiation of DNA replication.

Amino Acid Sequence↗
PubMed1994

Block of neuronal apoptosis by a sustained increase of steady-state free Ca2+ concentration.

Programmed death is a ubiquitous feature of the development of the vertebrate nervous system. This death is prevented in vivo by trophic factors and by afferent input. Death of neurons can also be prevented in culture models of programmed death by trophic factors and by chronic depolarization with elevated concentrations of K+ in the culture medium. The latter effect is mediated by Ca2+ influx through voltage-gated channels and may prevent death by mimicking survival-promoting effects of naturally occurring electrical activity. Little is currently known about the mechanism by which either trophic factors or increased cytoplasmic Ca2+ promote survival.

Animals↗
PubMed1994

Effects of nerve growth factor on rat peritoneal mast cells. Survival promotion and immediate-early gene induction.

Purified rat peritoneal mast cells in vitro die over a period of 2-6 days in conventional serum-containing medium. As mast cells die, they become pyknotic and undergo DNA fragmentation suggestive of an apoptotic process. Treatment of in vitro mast cells with nerve growth factor (NGF) greatly retards and reduces the death of mast cells (EC50 approximately 1 nM), with no effect on mast cell proliferation. Other neurotrophins have no such effect. NGF also induces the immediate early genes c-fos and NGFI-A with a similar dose dependence. In contrast to the secretagogue activity of NGF, neither the survival-promoting effect nor immediate early gene induction requires lysophosphatidylserine. The ability of NGF to promote mast cell survival is cell density-dependent and appears to be primarily because of induction of the synthesis and/or secretion of an autocrine survival factor by stimulated mast cells. These results suggest that the previously observed effects of NGF on mast cell numbers in vivo may in part be because of enhanced survival and that NGF may be an important mediator of mast cell function in normal and pathological states.

Animals↗
PubMed1994

A reevaluation of risk of in utero exposure to lithium.

OBJECTIVE: To reevaluate the risk associated with in utero exposure to lithium. DATA SOURCES AND STUDY SELECTION: Data were obtained from all published studies, in multiple languages, referenced in MEDLINE, Toxline, and the Lithium Information Center databases. Unpublished studies were not included. The search terms were lithium, pregnancy, teratogen, abnormalities (drug induced), Ebstein's anomaly, and adverse effects. DATA EXTRACTION AND SYNTHESIS: In the 1970s a very strong association was suggested between maternal lithium treatment during pregnancy and Ebstein's anomaly of the heart in the offspring. The relative risk for Ebstein's anomaly among such children was estimated to be 400 on the basis of data collected from a registry of voluntarily submitted cases. More recent controlled epidemiologic studies have consistently shown a lower risk. No women who took lithium during pregnancy were found among four case-control studies of Ebstein's anomaly involving 25, 34, 59, and 89 affected children, respectively. In two cohort studies, risk ratios of 3.0 (95% confidence interval [CI], 1.2 to 7.7) and 1.5 (95% CI, 0.4 to 6.8) for all congenital anomalies have been observed. The risk ratios for cardiac malformations in these studies were 7.7 (95% CI, 1.5 to 41.2) and 1.2 (95% CI, 0.1 to 18.3), respectively. CONCLUSION: While initial information regarding the teratogenic risk of lithium treatment was derived from biased retrospective reports, more recent epidemiologic data indicate that the teratogenic risk of first-trimester lithium exposure is lower than previously suggested. The clinical management of women with bipolar disorder who have childbearing potential should be modified with this revised risk estimate.

Abnormalities, Drug-Induced↗
PubMed1994

Neurites can remain viable after destruction of the neuronal soma by programmed cell death (apoptosis).

During the development of the nervous system extensive programmed neuronal death occurs that is regulated by neurotrophic factors. Invariably, degeneration and death of the neuronal soma as a result of trophic factor deprivation is accompanied by concurrent degeneration of the neurites. By examining the degeneration of sympathetic neurons after deprivation of their physiological trophic factor nerve growth factor, we show that the "slow Wallerian degeneration" allele (Wld6) expressed by homozygous mutant C57BL/Ola mice alters the normal time course of programmed neuronal death by selectively and dramatically delaying the onset of neurite disintegration. In contrast, degenerative events affecting the neuronal soma are not altered: Atrophy of the soma, apoptotic disintegration of the nucleus, commitment to die, and loss of viability occur normally. The enucleate neurites remaining after death of the soma have an intact plasma membrane, are metabolically active, and require an active metabolism for physical integrity. We suggest that the degeneration of neurites during developmentally occurring neuronal death is controlled by events confined to the neurites and occurs autonomously from the neuronal soma. Furthermore, programmed neuronal death of the soma proceeds independent from any influence exerted by degenerating neurites.

Animals↗
PubMed1994

Infection of sympathetic and sensory neurones with herpes simplex virus does not elicit a shut-off of cellular protein synthesis: implications for viral latency and herpes vectors.

Infection of non-neuronal cell types with herpes simplex virus type 1 (HSV-1) results in the degradation of host mRNA (Kwong & Frenkel 1987) and a shutoff in host protein synthesis (Roizman et al. 1965). This effect is mediated by a virion associated protein that is encoded by the viral vhs gene (Read & Frenkel 1983). This virion host shutoff (VHS) helps regulate viral gene expression and promotes efficient viral replication during the lytic cycle (Kwong & Frenkel 1987). Cultured sympathetic and sensory neurones, in contrast to primary rat fibroblasts, PC-12 cells, and Vero cells, showed no reduction in protein synthesis following infection with HSV-1. The resistance of neurones to VHS may be important in allowing establishment of a latent infection. In addition, this finding has a favourable impact on the idea of using HSV as a vector to deliver foreign genes into neurones.

Animals↗
PubMed1994

The role of nerve growth factor in modulating herpes simplex virus reactivation in vivo.

The role of nerve growth factor (NGF) in the modulation of herpes simplex virus (HSV) latency and reactivation was investigated in a mouse model. To determine whether NGF depletion would reactivate latent virus, concentrated anti-NGF serum antibodies were administered intraperitoneally to latently infected mice for 9 consecutive days. To determine whether NGF given prophylactically could suppress UV-B-induced viral reactivation, mice were irradiated with UV-B while being treated with NGF using diverse regimes over a 4-day period. Following intraperitoneal administration of anti-NGF antibodies, viral shedding was detected in a small number (10%) of mice, but it was not possible to pharmacologically suppress UV-B-induced viral reactivation with NGF. It would appear, therefore, that HSV latency in neurons innervating the cornea can be sustained and disrupted by physiological factors independent of NGF levels.

Animals↗
PubMed1994

Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death.

Sympathetic neurons undergo RNA and protein synthesis-dependent programmed cell death when deprived of nerve growth factor. To test the hypothesis that neuronal programmed cell death is a consequence of conflicting growth signals which cause the inappropriate activation of cell cycle genes, we have analyzed cell cycle-related genes for their expression in postmitotic neurons. Surprisingly, many of these genes are expressed in neurons, although cdc2, cdk2, and cyclin A are not. During programmed cell death, the expression of most of these genes, including several cyclins and the Rb and p53 tumor suppressor genes, decreases similar to that of neuronal genes. In contrast, cyclin D1 expression is selectively induced in dying neurons. Cyclin D1 mRNA levels peak 15-20 hr after nerve growth factor withdrawal, concurrent with the time that neurons become committed to die. These results provide an extensive characterization of cell cycle gene expression in postmitotic neurons and provide the evidence for a gene induced during neuronal programmed cell death.

Animals↗
PubMed1994

Postnatal development of survival responsiveness in rat sympathetic neurons to leukemia inhibitory factor and ciliary neurotrophic factor.

Embryonic rat sympathetic neurons undergo programmed cell death upon NGF deprivation. We show that during postnatal development, these neurons acquire the ability to be supported in vitro by LIF and CNTF as well as NGF. LIF and CNTF do not promote the long-term survival of embryonic day 21 sympathetic neurons in vitro. However, after 5 days of culture in the presence of NGF, the majority of embryonic day 21 sympathetic neurons can be supported by either of these factors. Furthermore, postnatal day 6 sympathetic neurons can be immediately supported by LIF and CNTF, indicating that acquisition of survival responsiveness occurs in vivo as well as in vitro. During this period, neuronal expression of LIF and CNTF receptor mRNAs remains constant, suggesting that sympathetic neurons alter their responsiveness to LIF and CNTF by allowing additional intracellular signaling pathways to promote survival.

Animals↗
PubMed1994

Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.

We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.

Animals↗
PubMed1994

Fetal NGF augmentation preserves excess trigeminal ganglion cells and interrupts whisker-related pattern formation.

In the developing nervous system, precisely patterned connections result from mechanisms that remodel initially diffuse connections. For example, ocular dominance column formation depends upon activity-based competitive interactions. In the developing trigeminal (V) somatosensory system, injury to afferent inputs prevents somatotopic pattern formation; however, afferent impulse blockade does not. What establishes central V patterns remains unclear. As a first step in assessing the role of neurotrophins in naturally occurring death of V ganglion cells and whisker-related pattern formation, the consequences of prenatal NGF injections were evaluated. Fetal rats given NGF on both embryonic day (E) 15 and E18 had 36% more V ganglion cells than normal and lacked whisker-related patterns in the V brainstem complex at birth and through postnatal day 3, as determined by cytochrome oxidase histochemistry. Rats injected with NGF on E16 or on E18, or with vehicle had normal ganglion cell numbers and brainstem patterns. Animals injected with antibodies to NGF or an NGF receptor had reduced ganglion cell numbers and normal brainstem patterns. These findings suggest that naturally occurring cell death in the V ganglion is neurotrophically regulated and that this process impacts upon somatotopic pattern formation in the V brainstem complex. Results of anterograde tracing experiments in NGF-augmented animals suggest that pattern disruptions are due to an absence of whisker-related patterning in the central projections of V ganglion cells. Moreover, single primary afferent collaterals labeled by Neurobiotin injections in the V ganglion did not have widespread or unusually complex arbors. Thus, NGF may affect V pattern formation by preserving or inducing projections to brainstem regions that normally come to lack such projections, such as the spaces normally demarcating neighboring whisker primary afferent projections.

Animals↗
PubMed1994

Correcting the PiZ defect in the alpha 1-antitrypsin gene of human cells by targeted homologous recombination.

BACKGROUND: Approximately 1% of people in North America carry the PiZ defect in the gene encoding the serum protein alpha 1-antitrypsin (AAT). Homozygotes are subject to early onset emphysema and liver disease. Here we demonstrate a means of correcting this gene defect, a point mutation in chromosome 14, by targeted homologous recombination. EXPERIMENTAL DESIGN: To target recombination to the AAT locus in human cells we use a normal complementary DNA cleaved once approximately 400 base pairs upstream of the PiZ mutation site in exon V. After transfection, targeting is monitored using a polymerase chain reaction assay devised to uniquely amplify successful homologous recombinants. RESULTS: When excercised on human PiZZ GM2522 fibroblasts, targeting replaces exon V of the endogenous gene, containing the PiZ mutation, with the exon V counterpart of a normal complementary DNA, simultaneously displacing the mutated exon V away from its promoter. By reversing the targeting strategy and using human PiM hepatoma cells, we have been able to supplant a normal copy of the AAT gene with a mutated PiZ version. This has allowed us to culture human HepG2 cells expressing the pathologic phenotype of the PiZ defect. Immunocytologic staining demonstrates the presence of inclusions in the liver cells due to aggregation of the PiZ isoform encoded by the recombined AAT gene under control of the endogenous gene promoter. CONCLUSIONS: (a) Targeting to the AAT locus is observed, using the methods we have developed, at rates that render feasible the selection of targeted cells and isolation of clones bearing a corrected AAT gene. (b) Correction of the exon V PiZ mutation alone, regardless of an accompanying exon III mutation, is sufficient to prevent aggregation of the PiZ protein in human liver cells. (c) Targeted homologous recombination offers advantages in potential gene therapy to correct AAT defects.

Animals↗
PubMed1993

NGF augmentation rescues trigeminal ganglion and principalis neurons, but not brainstem or cortical whisker patterns, after infraorbital nerve injury at birth.

Prior studies indicate that neonatal nerve injury kills many trigeminal (V) first- and second-order cells, and interrupts pattern formation in the brainstem and cerebral cortex. Yet it is not known whether effects upon cell survival and pattern formation are causally related. To determine whether axotomized V ganglion cells can be rescued by an exogenous trophic agent, rats received 5 mg/kg of nerve growth factor (NGF) prior to, and every day after, infraorbital nerve section on the day of birth until sacrifice on postnatal day (PND) 1, 3, 5, 7, or 14. Other animals received identical lesions without NGF. Ganglion cell numbers were significantly reduced by PND1 in pups not given NGF, while NGF-treated rats displayed no significant cell loss through PND7. However, NGF did not permanently rescue V neurons because ganglion cell numbers were reliably reduced by PND14. Cell numbers in V nucleus principalis were reduced by PND1 in pups not given NGF, while NGF-treated animals displayed no cell loss through PND14. NGF's rescue of second-order cells is probably an indirect effect of NGF action upon V ganglion cells because, in other newborns, NGF failed to maintain principalis cells after direct lesion of the left V ganglion. To determine whether preventing cell death permits whisker-related pattern formation, other rats also received NGF prior to and after infraorbital nerve section at birth. After 3-14 days, patterns were assessed in the brainstem and cortex with cytochrome oxidase histochemistry and serotonin immunocytochemistry. Whisker-related patterns failed to develop as in cases not given NGF. These data indicate that communication with the periphery is necessary for the maintenance of central whisker-related patterns. They also suggest that V ganglion cells can be rescued, albeit temporarily, from rapid injury-induced death by NGF, thereby delaying injury-induced cell death in nucleus principalis. However, the mechanism(s) responsible for injury-induced pattern alterations in the developing V system remains to be elucidated.

Animals↗