Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 109 records · Page 6Linked to original sources

PubMed1997

Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

Animals↗

PubMed1997

Neurturin, a novel neurotrophic factor, is localized to mouse chromosome 17 and human chromosome 19p13.3.

Neurturin is a potent neurotrophic factor closely related to glial cell line-derived neurotrophic factor (GDNF, 40% amino acid sequence identity) and, like GDNF, can promote the survival of numerous neuronal populations including sympathetic, nodose, and dorsal root ganglion sensory neurons. Both neurturin and GDNF signal through the Ret tyrosine kinase and require a glycosylphosphatidylinositol-linked coreceptor. Mutations in Ret and GDNF cause intestinal aganglionosis and renal dysplasia. Activating Ret mutations also cause multiple endocrine neoplasia syndromes (MEN2A and MEN2B). We have isolated mouse and human genomic neurturin clones. The sequence for preproneurturin is encoded by two exons. Mouse and human clones have common intron/exon boundaries. We have used interspecific backcross analysis to localize neurturin to mouse chromosome 17 near the Vav locus and fluorescence in situ hybridization analysis to localize human neurturin to the syntenic region of human chromosome 19p13.3.

Amino Acid Sequence↗

PubMed1997

Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons.

Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.

Animals↗

PubMed1997

Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway.

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.

Cell Line↗

PubMed1997

Inhibition of phosphatidylinositol 3-kinase activity blocks depolarization- and insulin-like growth factor I-mediated survival of cerebellar granule cells.

Depolarizing concentrations of potassium promote the survival of many neuronal cell types including cerebellar granule cells. To begin to understand the intracellular mediators of neuronal survival, we have tested whether the survival-promoting effect of potassium depolarization on cerebellar granule cells is dependent on either mitogen-activated protein (MAP) kinase or phosphatidylinositol 3-kinase (PI-3-K) activity. In 7-day cerebellar granule cell cultures, potassium depolarization activated both MAP kinase and PI-3-K. Preventing the activation of MAP kinase with the MEK1 inhibitor PD98059 did not affect potassium saving. In contrast, the survival-promoting effect of 25 mM potassium was negated by the addition of 30 microM LY 294002 or 1 microM wortmannin, two distinct inhibitors of PI-3-K. The cell death induced by PI-3-K inhibition was indistinguishable from the cell death caused by potassium deprivation; LY 294002-induced death included nuclear condensation, was blocked by cycloheximide, and had the same time course as potassium deprivation-induced cell death. Cerebellar granule cells can also be maintained in serum-free medium containing either 100 ng/ml insulin-like growth factor I (IGF-I) or 800 microM cAMP. PI-3-K inhibition completely blocked the survival-promoting activity of IGF-I, but had no effect on cAMP-mediated survival. These data indicate that the survival-promoting effects of depolarization and IGF-I, but not cAMP, require PI-3-K activity.

Androstadienes↗

PubMed1997

Surgical management of ventricular tachycardia.

BACKGROUND: Ventricular tachyarrhythmias are the leading cause of death from coronary artery disease. A small percentage of these arrhythmias originate in chronically ischemic myocardium, rather than acutely ischemic myocardium, and can be refractory to medical management. Epicardial mapping and focal cryoablation of foci demonstrating early activation may provide definitive therapy when pharmacologic management fails. We report a series of 42 consecutive patients with refractory ventricular tachycardia (VT) who were treated with open epicardial mapping and focal cryoablation after pharmacologic management failed. METHODS: We retrospectively reviewed the records of patients who underwent surgical treatment of malignant VT. For patients not recently seen in the clinic, we conducted telephone interviews. At the time of operation, epicardial mapping was performed to locate foci of early electrical activation. These foci were then cryoablated, using 2-minute applications of liquid nitrogen-cooled probes. All patients underwent postoperative electrophysiologic studies to test for inducible VT. RESULTS: Of these 42 patients, 34 (81%) were male, 8 (19%) female. Average age was 62.9 +/- 10.6 years; ejection fraction, 0.20 (range, 0.04 to 0.50); and number of foci ablated, 2.1 +/- 1.1 (range, 1 to 6). At the time of cryoablation, all patients underwent additional procedures, including aneurysmectomy, coronary artery bypass, or valve replacement. The 30-day operative mortality was 9.5% (4 of 42). Of the 38 survivors, 36 (94.7%) were clinically free of VT; the remaining 2 had spontaneous or inducible VT. CONCLUSIONS: Open cryoablation of foci propagating VT appears to be safe and effective. It may be the most definitive treatment for malignant VT.

Aged↗

PubMed1997

Disseminated infection due to Cylindrocarpon lichenicola in a patient with acute myeloid leukaemia.

We describe what is to our knowledge the first reported case of disseminated infection due to Cylindrocarpon. The presumed source was athlete's foot, a condition with which this fungus has previously been associated. Diagnosis was made by needle aspiration of a cutaneous lesion. Radiographic evidence of pulmonary involvement was present. The infection resolved following marrow regeneration and treatment with amphotericin B. Correct identification of Cylindrocarpon may be useful in guiding antifungal therapy.

Aged↗

PubMed1997

A risk assessment of topical tretinoin as a potential human developmental toxin based on animal and comparative human data.

BACKGROUND: Although topically applied all-trans-retinoic acid (tretinoin) undergoes minimal absorption and adds negligibly to normal endogenous levels, its safety in humans is occasionally questioned because oral ingestion of retinoids at therapeutic levels is known to entail teratogenic risks. OBJECTIVE: To assess the actual potential for developmental toxicity from treatment with topical tretinoin. METHODS: Risk assessments were conducted on four known human developmental toxicants (valproic acid, methotrexate, thalidomide, and isotretinoin) and a potential developmental toxicant (acetylsalicylic acid). The margin of safety for each chemical was calculated from the ratio of animal no-observed adverse effect levels to human lowest-observed adverse effect levels or estimated exposure doses. RESULTS: The derived safety margin of more than 100 for topical tretinoin (with 2% absorption) contrasted sharply with the near unity values for valproic acid, methotrexate, thalidomide, and isotretinoin and was larger than that for acetylsalicylic acid. CONCLUSION: These data support other epidemiologic and animal data that topical tretinoin is not a potential human developmental toxicant.

Abnormalities, Drug-Induced↗

PubMed1997

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

3T3 Cells↗

PubMed1997

Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection.

Given the increased choice of therapeutic agents and the rising incidence of serious invasive disease, it is important that reliable in-vitro methods for detecting antifungal drug resistance in Aspergillus spp. are developed. Six clinical isolates of Aspergillus fumigatus, obtained from patients in whom the clinical outcome was known, were selected for study. Each was used to examine a range of parameters affecting agar dilution and broth microdilution susceptibility test results. The in-vitro results were compared with outcome in a neutropenic mouse model of invasive aspergillosis. Groups of animals were treated with itraconazole at 25 mg/kg and 75 mg/kg and survival rates and organ burdens were determined. Itraconazole was efficacious against four isolates (susceptible) but failed for two (resistant) in the animal model of infection. Both the resistant isolates had been obtained from patients receiving itraconazole treatment with good serum concentrations of the drug. Conditions for the agar dilution test which produced results that correlated best with our in-vivo observations included the use of RPMI agar with L-glutamine buffered to pH 7 with MOPS, inoculated with 10(6)-10(7) conidia/mL and incubated for 48-72 h at 28 or 35 degrees C with a no-growth endpoint. Optimal conditions for the broth microdilution method included the use of RPMI medium with L-glutamine and 2% glucose buffered to pH 7 with MOPS, an inoculum of 2 x 10(5) conidia in 200 microL incubated for 48 h at 35 degrees C with a growth (or trace) endpoint. The MICs for the susceptible isolates were 0.12-1.0 mg/L and > or = 16 mg/L for the resistant isolates. With careful selection and standardization of test conditions it is possible to generate reproducible in-vitro susceptibility data for Aspergillus spp. that will predict clinical outcome.

Animals↗

PubMed1997

Programmed cell death in neurons: focus on the pathway of nerve growth factor deprivation-induced death of sympathetic neurons.

Extensive programmed cell death (PCD) occurs in the developing nervous system. Neuronal death occurs, at least in part, because neurons are produced in excess during development and compete with each other for the limited amounts of the survival-promoting trophic factors secreted by target tissues. Neuronal death is apoptotic and utilizes components that are conserved in other PCD pathways. In this review, we discuss the mechanism of trophic factor-dependent neuronal cell death by focusing on the pathway of nerve growth factor (NGF) deprivation-induced sympathetic neuronal death. We describe the biochemical and genetic events that occur in NGF-deprived sympathetic neurons undergoing PCD. Participation of the Bcl-2 family of proteins and the interleukin-1beta-converting enzyme family of proteases (caspases) in this and other models of neuronal death is also examined. The order and importance of these components during NGF deprivation-induced sympathetic neuronal death are discussed.

Animals↗

PubMed1997

In vitro studies of activity of voriconazole (UK-109,496), a new triazole antifungal agent, against emerging and less-common mold pathogens.

The in vitro activity of voriconazole was compared with that of itraconazole. Eighty-six isolates of pathogenic molds belonging to 23 species were tested by an agar dilution method in High Resolution medium. Voriconazole was more active than itraconazole against a number of hyaline molds, including several Fusarium spp. and Scedosporium prolificans. Voriconazole and itraconazole showed comparable good activity against several hyaline molds, including Penicillium marneffei and Scedosporium apiospermum, and a number of dematiaceous molds, including Bipolaris australiensis, Cladophialophora bantiana, several Exophiala spp., and several Fonsecaea spp. Our results suggest that voriconazole could be effective against a wide range of mold infections in humans.

Antifungal Agents↗

PubMed1997

Nerve growth factor antibody stimulates reactivation of ocular herpes simplex virus type 1 in latently infected rabbits.

Anti-nerve growth factor (anti-NGF) antibody has been shown to induce reactivation of latent herpes simplex virus type 1 (HSV-1) in vitro. We found that systemically administered anti-NGF induces ocular shedding of HSV-1 in vivo in rabbits harboring latent virus. Rabbits in which HSV-1 latency had been established were given intravenous injections of goat anti-NGF serum daily for 10 days beginning 42 days after primary viral infection. Tears were assayed for virus for 12 days beginning on the day of the first injection. All eight rabbits given high titer anti-NGF had infectious virus in their tears at least once during the 12-day period. Fifteen of 16 eyes were positive and the average duration of viral shedding for these eyes was 4.0 days. Latently infected rabbits receiving daily injections of nonimmune goat serum or saline for 10 consecutive days were controls. Only six of the 16 (38%) eyes from rabbits receiving nonimmune goat serum shed virus. Only one of 12 eyes from untreated rabbits shed virus. Sera from control rabbits had no detectable anti-NGF activity; titers in anti-NGF-treated rabbits ranged between 1:1000 and 1:10,000. NGF deprivation may act as a neuronal stressor and may share a common second messenger pathway with heat- or cold-stress induced reactivation of latent HSV-1.

Animals↗

PubMed1997

Effect of early cyclosporine levels on kidney allograft rejection.

Acute rejection is the greatest risk factor for development of biopsy-proven chronic rejection and late kidney allograft loss. We previously noted that low cyclosporine (CsA) levels were a risk factor for early acute rejection in pediatric recipients (1). In our current study, we used logistic regression to identify risk factors for acute rejection in 726 adult kidney transplant recipients on triple therapy (prednisone, azathioprine, CsA). Variables considered for logistic regression analysis were donor organ source (cadaver vs. living), degree of HLA mismatch (1 to 6 vs. 0 antigen mismatch), transplant number (primary vs. retransplant), CsA levels (< 125 vs. > or = 125 ng/ml, < 150 ng/ml vs. > or = 150 ng/ml, and < 175 vs. > or = 175 ng/ml), and acute rejection episodes (0 vs. > or = 1). Of 726 recipients, 401 (55%) received cadaver kidneys; 325 (45%), living related. Overall, 572 (79%) had a primary transplant; 154 (21%), a retransplant. The vast majority of acute rejection episodes occurred within the first 2 months posttransplant; 68% of recipients had no acute rejection episodes by 2 months and 58% had none by 60 months posttransplant. Logistic regression analysis revealed that a cadaver donor kidney (vs. living) (p = 0.004), a 1 to 6 antigen mismatch (vs. 0 mismatch) (p = 0.001), and CsA levels < 150 ng/ml (vs. > or = 150 ng/ml) correlated with biopsy-proven acute rejection. The correlation for CsA levels < 150 ng/ml (vs. > or = 150 ng/ml) held true for levels at 1 wk (p < 0.05), 1 month (p = 0.0001), 2 months (p = 0.01), and 3 months (p = 0.02) posttransplant. Similar correlation was found for CsA levels < 125 ng/ml (vs. > or = 125 ng/ml) and < 175 ng/ml (vs. > or = 175 ng/ml). Comparative analyses were made (by Chi-square) of acute and chronic rejection rates when recipients were divided into 3 groups by CsA level (< 125 ng/ml, > or = 125 to < 150 ng/ml, and > or 150 ng/ml). At each time point (1 wk, 2 wk, 1 month, 2 months, 3 months), CsA levels < 125 ng/ml (vs. > or = 125 to < 150 ng/ml and > or = 150 ng/ml) were associated with the greatest increased risk of acute rejection--for both cadaver and living related recipients (all p < 0.05). CsA levels < 125 ng/ml at each time point (1 wk, 2 wk, 1 month, 2 months, 3 months) were also associated with a significantly increased risk of chronic rejection (all p < 0.001). The incidence of both acute and chronic rejection was reduced in the group with CsA levels > or = 125 to < 150 ng/ml and further reduced in the > or = 150 ng/ml group. Our data indicate that maintaining CsA levels > or = 150 ng/ml as part of triple therapy reduces the incidence of both acute and chronic rejection. Because chronic rejection is the leading cause of late allograft loss, maintaining adequate CsA levels should improve long-term graft survival. Based on this analysis, we have modified our own immunosuppressive regimens to achieve higher CsA levels earlier posttransplant.

Adult↗

PubMed1997

Effect of initial slow graft function on renal allograft rejection and survival.

Cadaver renal allografts with immediate excellent function have good long-term outcomes, while grafts with delayed function have been associated with an increased incidence of acute rejection (AR) and subsequent poor long-term graft survival. There is, however, an intermediate group with initial slow function whose outcome is not well defined. This group was examined by reviewing 510 patients that received primary cadaver transplants between 1/85 and 8/95. Recipients were grouped by initial function into: 1) those with immediate graft function (IGF), defined by serum creatinine (Cr) level < 3 mg/dl on post-operative day (POD) 5 (n = 237); 2) those with slow graft function (SGF), defined by serum Cr > 3 mg/dl on POD 5 but no need for dialysis (n = 160); and 3) those with delayed graft function (DGF), defined by the need for dialysis in the first week post-transplant (n = 113). Five-year graft survivals were determined for each group by Kaplan-Meier methods and compared by a generalized Wilcoxon test. The incidence of AR in the first 6 months was significantly higher in those with SGF (40%) vs. those with IGF (30%) (p < 0.05); both groups had a lower incidence than those with DGF (47%) (p < 0.05). In the absence of AR, 5-yr graft survival was similar between the 3 groups, 94%, 97% and 92% for IGF, SGF and DGF respectively. In the presence of AR, 5-yr graft survival was significantly reduced in all groups, but most notably in those with SGF (51%) or DGF (53%), as compared to those with IGF (80%), (p < 0.001). We conclude that recipients with SGF, but no AR, have excellent outcomes, comparable to those with IGF. However, there is an increased incidence of early AR associated with SGF. In recipients with SGF or DGF, AR has a more profound detrimental effect on long-term graft survival than in the IGF group. Thus, recipients with SGF are at increased risk for AR with resultant poor long-term graft survival, and may need modified immunosuppressive protocols.

Adult↗

PubMed1997

Living kidney donation: donor risks and quality of life.

This review describes the immediate and long-term risks to kidney donors. We reviewed their perioperative morbidity and mortality as well as their quality of life after donation. The overall mortality in our series was zero. Nationally, donor mortality has been estimated to be 0.03% (5). Our overall complication rate was 8.2% with only 2 (0.2%) complications considered to be major (16). Complications were associated with male sex, body weight > or = 100 kg, and inadvertent entry into the pleura during the donor operation. Most of our donors were discharged from the hospital in < 5 days. Risk factors for a longer hospital stay were age 50 or older and an operative time of 4 hours or more. The average donor quality of life after donation, as measured by the SF-36, was better than that of the general US population. This finding persisted for years after donation. The vast majority of our donors found the experience to be very rewarding and would readily donate again if it were possible. However, 4% were dissatisfied and regretted their decision to donate a kidney; these were most likely to be donors other than a first-degree relative and donors whose recipient died within the first posttransplant year. Living donation of kidneys appears to be relatively safe, with very few physical and psychologic complications. It may even improve the donor's quality of life. Living donors are an underutilized source of kidneys. We continue to advocate and encourage living kidney donation.

Adult↗

PubMed1996

Chrysotile asbestos fibers mediate homologous recombination in Rat2 lambda fibroblasts: implications for carcinogenesis.

Asbestos fibers are widespread environmental carcinogens whose mutagenicity is now established. Nonetheless, the molecular nature of these mutations and the mechanisms by which they accelerate carcinogenesis remain poorly understood. We have assessed the ability of asbestos fibers to promote homologous recombination, a potent mechanism for generating intrachromosomal rearrangements, such as deletions, and mitotic recombination. For this, we have developed a new assay which determines the extent to which a marker gene present in DNA introduced by asbestos can recombine with homologous genes residing in a transfected cell. We have demonstrated that Calidria chrysotile fibers are mutagenic and are able to mediate transfection of molecularly marked mutant lacI genes in a manner that results in their preferential recombination with homologous wild-type genes in the transfected cell. Asbestos induced recombination events may play a significant role in asbestos mutagenesis and carcinogenesis, and promotion of recombination may underlie the well-recognized synergy of asbestos with other carcinogens.

Animals↗

PubMed1996

Neurturin, a relative of glial-cell-line-derived neurotrophic factor.

The normal development of the vertebrate nervous system entails the death of 30-70% of the neurons originally generated in most neuronal populations. This naturally occurring cell death is regulated by specific neurotrophic factors that promote neuronal survival and which are produced in limiting quantities by target cells, glial cells and neurons. These factors are also of potential utility as therapeutic agents for neurodegenerative diseases. Here we describe the purification and cloning of a new neurotrophic factor, identified on the basis of its ability to support the survival of sympathetic neurons in culture. This factor, neurturin, is structurally related to glial-cell-line-derived neurotrophic factor (GDNF). These factors can each activate the MAP kinase signalling pathway in cultured sympathetic neurons and support the survival of sympathetic neurons, as well as of sensory neurons of the nodose and dorsal root ganglia. Thus, neurturin and GDNF together now define a new family of neurotrophic factors.

3T3 Cells↗