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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 37 records · Page 2Linked to original sources
PubMed2001

RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons.

Sympathetic axons use blood vessels as an intermediate path to reach their final target tissues. The initial contact between differentiating sympathetic neurons and blood vessels occurs following the primary sympathetic chain formation, where precursors of sympathetic neurons migrate and project axons along or toward blood vessels. We demonstrate that, in Ret-deficient mice, neuronal precursors throughout the entire sympathetic nervous system fail to migrate and project axons properly. These primary deficits lead to mis-routing of sympathetic nerve trunks and accelerated cell death of sympathetic neurons later in development. Artemin is expressed in blood vessels during periods of early sympathetic differentiation, and can promote and attract axonal growth of the sympathetic ganglion in vitro. This analysis identifies RET and artemin as central regulators of early sympathetic innervation.

Animals↗
PubMed2000

Phosphatidylinositol 3-kinase is required for the trophic, but not the survival-promoting, actions of NGF on sympathetic neurons.

Nerve growth factor (NGF) supports target-dependent survival of sympathetic and other neurons during development; however, the NGF-regulated signaling pathways required for survival are not fully understood. Sympathetic neurons are able to abort acutely the cell death pathway initiated by NGF deprivation at early, as well as late, time points after readdition of NGF. We found that NGF-dependent phosphatidylinositol 3-kinase (PI-3-K) activity inhibited an early cell death event proximal to c-Jun phosphorylation. However, PI-3-K activity was not required for NGF to inhibit the translocation of Bax from the cytoplasm to the mitochondria, nor was it required for NGF to inhibit the subsequent release of mitochondrial cytochrome c, two events required for NGF deprivation-induced apoptosis. MEK/MAPK activity did not account for any of these NGF-dependent events. When subjected to long-term PI-3-K inhibition in the presence of NGF, the majority of sympathetic neurons did not die. Those that did die exhibited significant differences in the characteristics of death caused by PI-3-K inhibition as compared with NGF deprivation. Additionally, PI-3-K inhibition in the presence of NGF did not induce release of mitochondrial cytochrome c, indicating that these neurons were unable to complete the apoptotic program. In contrast to its modest effects on survival, inhibition of PI-3-K induced marked decreases in somal diameter and metabolic function, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, suggesting that PI-3-K is required for the trophic effects of NGF. Therefore, although PI-3-K is important for the trophic effects of NGF, it is not required for survival. Other, or at least additional, signaling pathways contribute to NGF-mediated survival of sympathetic neurons.

Animals↗
PubMed2000

Puralpha: a multifunctional single-stranded DNA- and RNA-binding protein.

Puralpha is a ubiquitous, sequence-specific DNA- and RNA-binding protein which is highly conserved in eukaryotic cells. Puralpha has been implicated in diverse cellular functions, including transcriptional activation and repression, translation and cell growth. Moreover, this protein has been shown to be involved in regulating several human viruses which replicate in the central nervous system (CNS), including human immunodeficiency virus type I (HIV-1) and JC virus (JCV). Puralpha exerts part of its activity by interacting with cellular proteins, including pRb, E2F, cyclin A, Sp1 and members of the Y-box family of proteins, including YB-1 and MSY1, as well as viral proteins such as polyomavirus large T-antigen and HIV-1 Tat. The ability of Puralpha to interact with its target DNA sequence and to associate with several viral and cellular proteins is modulated by RNA. Puralpha has also been shown to be involved in cell growth and proliferation. Its association with pRb, E2F and cyclin A coupled with its fluctuating levels throughout the cell cycle, position Puralpha as a crucial factor in the cell cycle. Moreover, microinjection studies demonstrate that Puralpha causes either a G(1) or G(2) arrest depending on the cell cycle time of injection. The gene encoding Puralpha has been localized to a human locus which is frequently deleted in myelogenous leukemias and other cancers and Puralpha gene deletions have been detected in many cases of lymphoid cancers. The following review details the structural characteristics of Puralpha, its family members and the involvement of this protein in regulating various cellular and viral genes, viral replication and cell growth.

Amino Acid Sequence↗
PubMed2000

Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization.

Nerve growth factor (NGF) deprivation induces a Bax-dependent, caspase-dependent programmed cell death in sympathetic neurons. We examined whether the release of cytochrome c was accompanied by the loss of mitochondrial membrane potential during sympathetic neuronal death. NGF- deprived, caspase inhibitor-treated mouse sympathetic neurons maintained mitochondrial membrane potential for 25-30 h after releasing cytochrome c. NGF- deprived sympathetic neurons became committed to die, as measured by the inability of cells to be rescued by NGF readdition, at the time of cytochrome c release. In the presence of caspase inhibitor, however, this commitment to death was extended beyond the point of cytochrome c release, but only up to the subsequent point of mitochondrial membrane potential loss. Caspase-9 deficiency also arrested NGF-deprived sympathetic neurons after release of cytochrome c, and permitted these neurons to be rescued with NGF readdition. Commitment to death in the NGF-deprived, caspase- 9-deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factor-deprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Inhibition of apoptotic signaling cascades causes loss of trophic factor dependence during neuronal maturation.

During development, neurons are acutely dependent on target-derived trophic factors for survival. This dependence on trophic support decreases dramatically with maturation in several neuronal populations, including sympathetic neurons. Analyses of nerve growth factor deprivation in immature and mature sympathetic neurons indicate that maturation aborts the cell death pathway at a point that is mechanistically indistinguishable from Bax deletion. However, neither the mRNA nor protein level of BAX changes with neuronal maturation. Therefore, BAX must be regulated posttranslationally in mature neurons. Nerve growth factor deprivation in immature sympathetic neurons induces two parallel processes: (a) a protein synthesis-dependent, caspase-independent translocation of BAX from the cytosol to mitochondria, followed by mitochondrial membrane integration and loss of cytochrome c; and (b) the development of competence-to-die, which requires neither macromolecular synthesis nor BAX expression. Activation of both signaling pathways is required for caspase activation and apoptosis in immature sympathetic neurons. In contrast, nerve growth factor withdrawal in mature sympathetic neurons did not induce the translocation of either BAX or cytochrome c. Moreover, mature neurons did not develop competence-to-die with cytoplasmic accumulation of cytochrome c. Therefore, inhibition of both BAX-dependent cytochrome c release and the development of competence-to-die contributed to the loss of trophic factor dependence associated with neuronal maturation.

Animals↗
PubMed2000

Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRalpha1 RET-specific agonists.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.

3T3 Cells↗
PubMed2000

Interaction of HIV-1 Tat with Puralpha in nuclei of human glial cells: characterization of RNA-mediated protein-protein binding.

A complex between the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), and the cellular protein, Puralpha, has been implicated in activation of the late promoter of JC virus (JCV) and in enhancement of JCV DNA replication. JCV is the causative agent of progressive multifocal leukoencephalopathy (PML), an acquired immunodeficiency syndrome (AIDS) opportunistic infection of the brain. Puralpha also binds the HIV-1 TAR RNA element and activates HIV-1 transcription, suggesting a role for RNA binding in the action of this protein. Using immunoelectron microscopy, we find that in human glial cells expressing both proteins, Tat and Puralpha are colocalized in extranucleolar chromatin structural elements. The colocalized Puralpha and Tat are nearly exclusively nuclear, although individual proteins can be seen in both nucleus and cytoplasm, suggesting a preferential tropism of the complex for the nucleus. Analysis of the interaction between purified proteins indicates that the Tat-Puralpha interaction is strongly enhanced by the presence of RNA. Tat amino acids from 37-48 are essential for Tat binding. Residues 49-72, including the TAR RNA-binding domain, are critical for binding to Puralpha, while Cys(22), in the Tat transactivation domain, is responsible for an important global effect. Puralpha repeat II domains are involved in the interaction, and a polypeptide based on one such sequence inhibits binding. After RNase treatment of Puralpha enhancement of Tat binding can be partially restored by addition of a single-stranded JCV DNA PUR element, to which Tat does not bind. The results indicate that the Tat-Puralpha interaction is direct, rather than through an RNA link, and that RNA binding configures Puralpha for optimal interaction with Tat.

Biological Transport↗
PubMed2000

Reversible physiological alterations in sympathetic neurons deprived of NGF but protected from apoptosis by caspase inhibition or Bax deletion.

Cell death in nervous system development and in many neurodegenerative diseases appears to be apoptotic or programmed. Withdrawal of nerve growth factor (NGF) from cultures of superior cervical ganglia neurons (SCG) is an excellent model of programmed cell death (PCD), producing apoptosis within 24-48 h. This death can be prevented by treatment with caspase inhibitors or deletion of the proapoptotic Bax gene. Since inhibition of apoptosis is an attractive strategy for the therapy of many neurological diseases and little is known about the function of neurons when apoptosis has been aborted, we examined the electrophysiological properties of NGF-deprived SCG neurons from rats and mice, saved by the caspase inhibitor boc-aspartyl(OMe)fluoromethyl ketone (BAF) or by Bax deletion. Compared to NGF-maintained controls, the resting membrane potentials of BAF-saved neurons were depolarized by 9 mV and the action potentials were prolonged by over 50%. Nicotinic cholinergic current density was depressed by about 50%. Electrophysiological parameters returned to normal within 4 days after NGF restoration. Neurons from Bax-deficient mice were altered differently by NGF withdrawal. There were no detectable changes in resting or action potentials. However, nicotinic current density was reduced just as in BAF-saved rat neurons. There were no observable changes in the processes of individual neurons after 6 days of NGF deprivation in the presence of BAF. Our results indicate that neurons are physiologically altered during pharmacological inhibition of PCD, but fully recover after trophic support is returned.

Amino Acid Chloromethyl Ketones↗
PubMed2000

GFRalpha-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival.

The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.

Animals↗
PubMed2000

The GDNF family ligands and receptors - implications for neural development.

The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system.

Animals↗
PubMed2000

Staurosporine-induced neuronal death: multiple mechanisms and methodological implications.

To examine whether multiple pathways of cell death exist in sympathetic neurons, we studied the cell death pathway induced by staurosporine (STS) in sympathetic neurons and compared it with the well-characterized NGF deprivation-induced death pathway. Increasing concentrations of STS were found to induce sympathetic neuronal death with different biochemical and morphological characteristics. One hundred nM STS induced metabolic changes, loss of cytochrome c, and caspase-dependent morphological degeneration which closely resembled the apoptotic death induced by NGF deprivation. In contrast, sympathetic neurons treated with 1 microM STS showed no loss of cytochrome c but exhibited extensive, caspase-independent, chromatin changes that were not TUNEL positive. One microM STS-treated sympathetic neurons had greatly reduced metabolic activities and became committed to die rapidly, yet maintained soma structure and appeared viable by other criteria even up to 48 h after STS treatment, illustrating the need to assess cell death by multiple criteria. Lastly, in contrast to the cell death-inducing activities of 100 nM STS or 1 microM STS, very low concentrations of STS (1 nM STS) inhibited sympathetic neuronal death by acting either at or prior to c-jun phosphorylation in the NGF deprivation-induced PCD pathway.

Animals↗
PubMed2000

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Animals↗
PubMed2000

Salvage therapy of open, infected surgical wounds: a retrospective review using Techni-Care.

OBJECTIVE: To determine outcome of infected surgical wounds treated with 3% para-chloro-meta-xylenol + 3% phospholipid PTC [PCMX-PL] (Techni-Care). DESIGN: Retrospective review of patient records. SETTING: University hospital. PATIENTS: Thirty consecutively treated patients (sixteen male, fourteen female) who had developed open infected wounds (twenty-one abdominal [seventy percent], nine extremity [thirty percent]). Mean patient age was 50.1 years. All wounds were treated with commonly practiced wound care techniques (e.g., debridement, frequent dressing changes using saline or topical antibiotics, and, in most cases, parenteral antibiotics) for an extended period of time prior to intervention (mean = 35 days). INTERVENTIONS: PCMX-PL, a topical microbicide, was used as adjunctive therapy. Eight outcome parameters were analyzed: (1) patient morbidity and mortality; (2) wound healing; (3) number of debridements; (4) wound culture results; (5) leukocytosis (peripheral white blood cell count > 10,000 cells/microl); (6) number of febrile days (temperature > 101 degrees F); (7) length of hospital stay; and (8) number of days of intensive care. RESULTS: No treatment failures or adverse reactions to PCMX-PL were seen. Twenty (sixty-seven percent) wounds were healed or had been successfully closed while ten (thirty-three percent) were granulating well at sixty-day follow-up. The number of debridements, positive wound cultures, white-blood-cells, and febrile days decreased after PCMX-PL treatment began. CONCLUSIONS: Despite severe underlying diseases, all patients were discharged from the hospital with closed or healing wounds. We recommend treatment with PCMX-PL as an adjunctive therapy for infected wounds particularly when standard care measures have failed.

Adult↗
PubMed2000

Use of herbal therapies by adults seen in an ambulatory care research setting: an exploratory survey.

OBJECTIVE: To identify and characterize patterns of use of herbal products among patients participating in selected research clinics. DESIGN: Survey of three National Institutes of Health (NIH) ambulatory care research clinics. SUBJECTS: Convenience sample of 490 adult patients (168 male, 322 female) attending rheumatology, liver, and endocrinology/metabolic research clinics. RESULTS: Of the patients surveyed, 16.7%: (n = 82) reported using herbs. There were no significant sociodemographic differences between herb and nonherb users. Indications for herb use differed among the disease groups; patients in the endocrine and rheumatology clinics were taking herbs predominantly for "energy" or "wellness"; those attending the liver clinic tended to use herbal therapies as treatment for their disease. Mean and median monthly expenditure for herbal products was $30 and $10, respectively. There was a significant positive correlation between number of herbs used and use of other dietary supplements (p < 0.0001). CONCLUSIONS: One in six patients in ambulatory clinical research settings may be taking herbal products in addition to prescribed treatment. This figure is lower than in the general population, possibly because the patients may stop using herbs when participating in a research project. Although empirical evidence on the beneficial or adverse effects of herb therapy alone or in combination with drug therapies is limited, clinical researchers should be aware of the potential for confounding clinical trial results.

Adult↗
PubMed2000

Lack of correlation of in vitro amphotericin B susceptibility testing with outcome in a murine model of Aspergillus infection.

Amphotericin B has been the standard therapy for invasive aspergillosis since its introduction in 1957. It is only moderately effective. Many susceptibility tests have been used but little variation has been noted between strains. We have studied three strains of Aspergillus fumigatusand one of Aspergillus terreusin a neutropenic mouse model of invasive aspergillosis and attempted to correlate the variable efficacy in vivowith MICs generated by over 30 different susceptibility test formats. One strain of A. fumigatus(AF65) and the strain of A. terreus(AT49) were 'resistant' and the remaining two strains of A. fumigatus(AF210 and AF294) were 'susceptible' in vivo. Only AT49 had elevated MICs of amphotericin (MIC 2 mg/L) by 41 of 54 in vitrotesting systems. With each test format, including Etest, there was no distinction between MICs obtained for AF65, AF210 and AF294 (MICs 0.125-64 mg/L depending on the test). Thus despite extensive efforts we have been unable to correlate susceptible test results with in vivooutcome in A. fumigatusbut we have with A. terreus, with some test formats. This suggests that, at present, amphotericin B susceptibility testing of A. fumigatus is of limited clinical value and further work needs to be done to find testing systems that can identify the 'resistance' documented in vivo.

Amphotericin B↗
PubMed2000

Developmental toxicity studies in rats and rabbits with 3,5,6-trichloro-2-pyridinol, the major metabolite of chlorpyrifos.

3,5,6-Trichloro-2-pyridinol (TCP), the primary metabolite of chlorpyrifos and chlorpyrifos-methyl, was evaluated for potential developmental toxicity. Groups of 32-34 bred female Fischer 344 rats were given 0, 50, 100, or 150 mg TCP/kg/day by gavage on gestation days 6-15; the fetuses were evaluated on gestation day 21. Similarly, groups of 16 inseminated female New Zealand White rabbits were given 0, 25, 100, or 250 mg TCP/kg/day by gavage on gestation days 7-19, and fetuses were evaluated on gestation day 28. No clinical signs of toxicity attributed to TCP were noted in either species. In rats, at 150 mg/kg/day, maternal effects included slight decreases in feed consumption, significantly depressed body weight gain (25% relative to controls) resulting in significantly lower maternal terminal body weights, and increased relative liver weight. At 100 mg/kg/day, maternal body weight gain in rats was depressed approximately 22%. Among rabbits, maternal effects were limited to the group given 250 mg/kg/day, which lost an average of approximately 70 g during the treatment period (vs. 140 g in the controls). There were no effects on fetal weight or viability, nor were there significant increases in any fetal alteration in either species. A slightly higher (not statistically significant) than usual incidence of central nervous system anomalies occurred in rabbits, but these anomalies were found in both treated and control groups in this study as well as contemporaneous studies of unrelated compounds. This, and the fact that these anomalies were not seen with the parent compound, chlorpyrifos, suggest that their origin was spontaneous. Thus, TCP was not considered fetotoxic or teratogenic in either rats or rabbits, even at dose levels that produced maternal toxicity.

Abnormalities, Drug-Induced↗
PubMed2000

Aspergillus antigen testing in bone marrow transplant recipients.

AIMS: To assess the clinical usefulness of a commercial aspergillus antigen enzyme linked immunosorbent assay (ELISA) in the diagnosis of invasive aspergillosis (IA) in bone marrow transplant recipients, and to compare it with a commercial latex agglutination (LA) test. METHODS: In total, 2026 serum samples from 104 bone marrow transplant recipients were tested. These comprised 67 sera from seven patients who had died with confirmed IA, 268 sera from nine patients who had died with suspected IA, and 1691 sera from 88 patients with no clinical, radiological, or microbiological signs of IA. RESULTS: The ELISA was more sensitive than the LA test. All patients who were ELISA positive were also LA positive, and a positive LA result never preceded a positive ELISA. Twelve of 16 patients with confirmed or suspected IA were ELISA positive on two or more occasions, compared with 10 of 15 who were LA positive. ELISA was positive before LA in five patients (range, 2-14 days), and became positive on the same day in the remainder. Aspergillus antigen was detected by ELISA a median of 15 days before death (range, 4-233). Clinical and/or radiological evidence of IA was noted in all patients, and a positive ELISA was never the sole criterion for introduction of antifungal treatment. Two samples (one from each of two patients without IA) gave false positive results. CONCLUSIONS: The aspergillus ELISA is a specific indicator of invasive aspergillosis if the criterion of two positive samples is required to confirm the diagnosis. However, the test is insufficiently sensitive to diagnose aspergillosis before other symptoms or signs are apparent, and hence is unlikely to lead to earlier initiation of antifungal treatment. It is therefore unsuitable for screening of asymptomatic patients at risk of invasive aspergillosis, but does have a useful role in confirming the diagnosis in symptomatic patients.

Adolescent↗