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Transforming growth factor beta within fibrotic scleroderma lungs.

PURPOSE, PATIENTS, AND METHODS: Since transforming growth factor beta (TGF beta) has been implicated as an important mediator of pulmonary fibrosis, we measured TGF beta protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGF beta protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms. RESULTS: Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGF beta 1 and immunoreacted with anti-TGF beta antibody. The normal population had a significantly higher average TGF beta concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGF beta 1 gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGF beta concentration variability did not correlate with variations in BAL cellularity or TGF beta 1 gene expression within the recovered mononuclear cells. CONCLUSIONS: It is concluded that both normal and fibrotic lungs have TGF beta 1 present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGF beta protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGF beta protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGF beta protein concentration at the alveolar epithelial surface.

Adult↗

Delineation of electric and magnetic field effects of extremely low frequency electromagnetic radiation on transcription.

The relative effects of the electric and magnetic field components of extremely low frequency electromagnetic radiation (ELF) on transcription were examined in human leukemia HL-60 cells. Delineation of the individual field contributions was achieved by irradiating cells in separate concentric compartments of a culture dish within a solenoid chamber. This exposure system produced a homogeneous magnetic field with a coincident electric field whose strength varied directly with distance from the center of the culture dish. Irradiation of HL-60 cells with sine wave ELF at 60 Hz and a field strength of 10 Gauss produced a transient increase in the transcriptional rates which reached a maximum of 50-60% enhancement at 30-120 minutes of irradiation and declined to near basal levels by 18 hours. Comparison of transcription responses to ELF of cells in different concentric compartments revealed that the transcriptional effects were primarily the result of the electric field component with little or no contribution from the magnetic field.

Cell Line↗

Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase.

The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.

Baculoviridae↗

Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Adult↗

A new mechanism for the toxicity of ozone.

Ozone, with its high reactivity, is entirely consumed as it passes through the first layer of tissue it contacts at the lung/air interface. This layer includes the epithelial cell lining fluid (ELF) and, where the ELF is thin or absent, the membranes of the epithelial cells that line the airways. Thus the biochemical changes that follow the inhalation of ozone must be relayed into deeper tissue strata by a cascade of ozonation products. Lipid ozonation products (LOP) are suggested to be the most likely relay molecules of ozone's signal. This is because unsaturated fatty acids are present in relatively high concentrations in both the ELF and in pulmonary cell bilayers, and ozone reacts with unsaturated fatty acids to produce ozone-specific products. Further, LOP are finite in number, have structures that are predictable from the Criegee ozonation mechanism, and are small, diffusible, stable (or meta-stable) molecules, similar to other lipid-derived signal transduction species. Preliminary data show that individual LOP cause the activation of specific lipases, which trigger the release of endogenous mediators of inflammation.

Animals↗

Embryonic expression of eph signalling factors in Xenopus.

Cellular communication in the developing embryo is mediated by receptor-ligand interactions at the cell surface. Receptor protein tyrosine kinases (RTKs) have been shown to play a critical role in the development of the vertebrate embryo. The eph receptors are a large subclass of RTKs for which a corresponding ligand family has only recently been described. The restricted expression patterns of several eph receptors imply roles for these molecules in early vertebrate development. We have isolated both a ligand of the eph ligand family (ELF), that we have named XELF-a, and an eph-related receptor, XE10, the likely homolog of the murine eck/Sek-2 receptor. At least two forms of the XELF-a transcript are present in the developing embryo. A truncated form of the XELF-a ligand, XELF-á, is the first ELF ligand isolated that lacks both the membrane-spanning and membrane-anchoring motifs conserved among this family, suggesting that ELF ligands can function as fully soluble molecules in vivo. XELF-a and XE10 are expressed maternally and throughout early embryogenesis, while XELF-á is only expressed zygotically. The dynamic expression patterns of these signalling molecules, in both mesoderm and neurectoderm, suggest that they may play a role in the patterning of the early vertebrate embryo.

Amino Acid Sequence↗

Alveolar lavage fluid (ALF) of normal volunteer subjects: cytologic, immunocytochemical, and biochemical reference values.

OBJECTIVE: Pooled bronchoalveolar lavage fluid (BALF), the return of lavage, contains both bronchial and alveolar material which differ from each other. Artifacts may be created by filtering, centrifuging and washing cells before cytopreparation. This study presents reference values of healthy volunteers for the alveolar sample, ALF, cytopreparation being performed without filtration or centrifugation. METHODS: Eighteen healthy, non-smoking volunteers underwent a standard bronchoalveolar lavage using 10 aliquots of 20 ml of saline. Excluding the return of the first and second aliquots, the rest were pooled and examined cytologically, immunocytochemically and biochemically. The mean, standard deviation, and 95% confidence limits were calculated for the following variables: amount of return, estimated content of epithelial lining fluid (ELF), total and differential cell counts on filter and cytocentrifuge (CCF) preparations, computed cell counts per unit volume of ALF, distribution of lymphocyte subgroups CD3+CD2, CD4, CD8, CD19, CD25 and CD57, and the ratio of CD4 to CD8, the amounts of lymphocytes in the same subgroups per volume of ALF, and the concentrations of total protein, albumin, immunoglobulins A, G and M, hyaluronic acid, eosinophilic cationic protein (ECP), procollagen III aminoterminal propeptide (PCP) and beta 2-microglobulin in ALF and in ELF, as well as the ratios of the concentrations of the solutes in ALF to the same in serum. RESULTS: The 95% confidence limits of means for the most important variables were as follows: estimated ELF content 0.42-0.74%; total cells in ALF 76.6-143.0 x 10(6) l-1; distribution of inflammatory cells on filter and CCF slides: macrophages 74.9-83.6 and 81.4-90.1%, lymphocytes 13.1-22.5 and 8.1-16.4%, and neutrophils 1.0-4.1 and 0.7-2.7%, respectively; distribution of lymphocyte subsets: CD3+CD2 85.6-90.6%, CD4 44.3-53.1%, CD8 26.9-35.8%; concentration of solutes in ALF: total protein 44.8-61.3 mg l-1, albumin 15.4-22.2 mg l-1, IgA 1.8-3.4 mg l-1, IgG 3.1-6.1 mg l-1, IgM 0.05-0.26 mg l-1, hyaluronic acid 8.8-11.1 micrograms l-1, ECP 0.19-0.77 micrograms l-1, PCP 0.005-0.58 micrograms l-1, beta 2-microglobulin 62.2-81.5 micrograms l-1. CONCLUSIONS: Our results show that excluding the bronchial sample from ALF of volunteer subjects and omitting filtering and washing before cytopreparation produces cytologic, immunocytochemical and biochemical reference values with reasonable 95% confidence limits to be used in clinical settings.

Adult↗

Lack of effect of extremely low frequency electromagnetic fields on cyclin-dependent kinase 4 inhibitor gene p18(INK4C) in electric energy workers.

BACKGROUND: Long-term exposure to extremely low frequency magnetic fields (ELF-MFs) may be a risk factor for human cancer. One mechanism through which ELF-MFs could influence neoplastic development is the deletion/mutation of cancer-related genes. Cellular proliferation follows an orderly progression through the cell cycle, which is governed by different cyclins and cyclin-dependent kinase inhibitors (CDKIs). The putative tumor suppressor gene p18(INK4C) encodes a specific inhibitor of cyclin D-cyclin-dependent kinase 4 inhibitor complexes having an important role in cell-cyclin regulation. It has been found to be deleted/mutated in a variety of human cancers. Therefore, this study is to investigate whether or not long-term extremely low frequency electromagnetic field exposure may be a risk factor for human cancer due to the gene p18(INK4C) deletion/mutation. METHODS: The study was carried out on 31 male electric workers and 30 healthy males between 30 and 40 years of age from the same geographic area and with similar lifestyles. We studied both groups by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: In comparison to the controls, band migration of exon 1 was found to be indifferent in all the subjects tested. However, only exon 2 of two electric workers was slow in migration with respect to both control and other subjects in the same class. This slow migration suggests that point mutations or polymorphisms may exist in this region of the p18(INK4C) gene. The relative risk (RR) for the unmatched analysis was 1,069 (95% confidence interval [CI] 0.975-1.172). CONCLUSIONS: The results suggest that long-term ELF-MFs exposure does not significantly increase the risk of cancer.

Adult↗

Survey of residential extremely-low-frequency magnetic field exposure among children in Taiwan.

This survey estimated the extremely-low-frequency magnetic field (ELF-MF) exposure in households with children under the age of 7 years in Taiwan. To select a representative sample, we used the probability proportional to size sampling technique and simple random cluster sampling technique to select study districts in each city/county of Taiwan. A total of 40 districts were selected and all households with children aged less than 7 (n=4184) were visited and solicited for in-home measurements. Four trained interviewers carried out spot-measurements in rooms of each selected household during a two-year period. A questionnaire inquiring residential characteristics and nearby power facilities was also administered to a parent or caregiver. ELF-MF measurements were performed in a total of 2214 households (53% participation rate). Controlling for participation rate, the weighted overall mean magnetic field exposure was estimated at 0.121 (standard deviation 0.185) micro-Tesla (microT) (range 0.010-3.304 microT). Mean exposures were higher than 0.3 and 0.4 microT in an estimated 7.3% (n=159) and 5.4% (n=115) of the households, respectively. There was a substantial agreement between overall mean residential exposure and exposure measured in children's bedrooms. In a multivariate logistic regression model, we noted that buildings with advanced age, buildings for both residential and commercial uses, and buildings close to power facilities were more likely a to have mean exposure above 0.4 microT. Our survey results indicate that residential ELF-MF exposure is likely to be higher for children in Taiwan than for children in Europe and North America.

Child↗

Antioxidant-mediated augmentation of ozone-induced membrane oxidation.

The pulmonary epithelial lining fluid (ELF) contains substrates, e.g., ascorbic acid (AH2), uric acid (UA), glutathione (GSH), proteins, and unsaturated lipids, which undergo facile reaction with inhaled ozone (O3). Reactions near the ELF gas/liquid interface likely provide the driving force for O3 absorption ("reactive absorption") and constrain O3 diffusion to the underlying epithelium. To investigate the potential mechanisms wherein O3/ELF interactions may induce cellular damage, we utilized a red cell membrane (RCM) model intermittently covered by an aqueous film to mimic the lung surface compartmentation, and evaluated exposure-mediated loss of acetylcholinesterase activity (AChE) and TBARS accumulation. In the absence of aqueous reactants, O3 exposure induced no detectable changes in AChE or TBARS. AH2 and GSH preferentially induced oxidative damage in a dose-dependent fashion. AH2-mediated RCM oxidation was not inhibited by superoxide dismutase, catalase, mannitol, or Fe chelators. O3 reaction with UA, Trolox, or albumin produced no RCM oxidation but oxidation occurred when AH2 was combined with UA or albumin. Rat bronchoalveolar lavage fluid (BALF) also induced RCM oxidation. However, in vivo O3 exposure dampened the extent of BALF-mediated RCM oxidation. Although we cannot completely rule out O3 diffusion to the RCM, product(s) derived from O3 + AH2/GSH reactions (possibly O3*- or 1O2) likely initiated RCM oxidation and may suggest that in vivo, such secondary species account for O3 permeation through the ELF leading to cellular perturbations.

Acetylcholinesterase↗

Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail Helix aspersa.

Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.

Animals↗

Using urea dilution to standardise cellular and non-cellular components of pleural and bronchoalveolar lavage (BAL) fluids in the cat.

A technique to standardise the analysis of cellular and non-cellular components in epithelial lining fluid (ELF) collected during saline lavage of pulmonary and pleural cavities was developed using the urea dilution method. Bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected from 12 clinically healthy cats. Total and differential cell counts in BAL fluid were within normal ranges for the cat, while cell counts in PL fluid were assumed to be normal based on clinical health during examination, auscultation and lactate dehydrogenase (LDH) activities being comparable with other species. The major clinical implication of this study was that nucleated cell counts within feline ELF could not be predicted from analysis of lavage fluid which suggests that calculation of the proportion of ELF in lavage fluid by the urea dilution method may be necessary to avoid misdiagnosis of health or disease in pulmonary or pleural cavities.

Animals↗

Non-thermal effects of power-line magnetic fields (50 Hz) on gene expression levels of pluripotent embryonic stem cells-the role of tumour suppressor p53.

The diffusion of extremely low-frequency (50 Hz) electromagnetic fields (ELF-EMF) in the human environment raises the question of the induction of biological effects of EMF on mammalian cells. We used the model of mouse pluripotent embryonic stem (ES) cells, which have the capacity to develop in vitro into cells of all lineages, to analyse non-thermal effects of ELF-EMF. Wild type (wt) and p53-deficient ES cells were exposed under controlled conditions to ELF-EMF signals simulating power-line (50 Hz) magnetic field (PL-MF) exposure. Different flux densities of 0.1 mT, 1.0 mT or 2.3 mT and intermittency schemes with various ON/OFF cycles were applied for 6 h or 48 h during the first stages of cell differentiation. Transcript levels of regulatory genes, such as egr-1, p21, c-jun, c-myc, hsp70 and bcl-2, were analysed by semi-quantitative RT-PCR immediately after exposure or after a recovery time of 18 h. Intermittent PL-MF exposure to 5 min ON/30 min OFF cycles at a flux density of 2.3 mT for 6 h resulted in a significant up-regulation of c-jun, p21 and egr-1 mRNA levels in p53-deficient, but not in wild-type cells. No significant effects were observed in both cell systems by PL-MF at lower flux densities, longer exposure time or after 18 h recovery time. Our data indicate that 5 min ON/30 min OFF intermittent PL-MF exposure is capable of evoking non-thermal responses in ES cells, dependent on the cellular p53 function. The nature of the biological responses triggered by PL-MF is discussed.

Animals↗

Exposure to extremely low-frequency electromagnetic fields improves social recognition in male rats.

The effect of exposure to low-frequency electromagnetic fields (ELF EMFs) on social recognition was studied. The test was based upon a comparison between two encounters of an adult rat and a conspecific juvenile, separated by an interexposure interval (IEI). The exposure to ELF EMF of 1 mT intensity during 2 h for 9 days increased the duration of short-term memory of adult male Wistar rats up to 300 min. These data indicate, for the first time, that ELF EMF improves social recognition memory in rats.

Animals↗

Weak electromagnetic fields (50 Hz) elicit a stress response in human cells.

The aim of this study was to demonstrate the expression of heat shock (HS) genes in human cells in response to extremely low-frequency electromagnetic fields (ELF-EMF) alone and in combination with thermal stress. After exposing human myeloid leukemia (HL-60) cells to the stressor(s) for 30 min we quantified the expression of the HS genes HSP27, HSP60, HSP70 (A, B, and C), HSC70, HSP75, HSP78, and HSP90 (alpha and beta) by RT-PCR. The results clearly show that HS genes, in particular the three HSP70 genes (A, B, and C), are induced by ELF-EMF, a reaction that is enhanced by simultaneous HS (43 degrees C for 30 min). The results show similarities and some significant differences to previous experiments in which transgenic nematodes were used to monitor the induction of the HSP70 gene under similar stress conditions. We also studied the effect of different flux densities on gene expression in the range of 10-140 microT. Even the lowest dose tested (10 microT) resulted in a significant induction of the genes HSP70A, HSP70B, and HSP70C. The reaction to ELF-EMF shows a maximum at a flux density of 60-80 microT. The unusual dose-response relation reveals an interesting difference to other stressors that elicit the HS response.

Acute Disease↗

Aerosol delivery and safety of recombinant human deoxyribonuclease in young children with cystic fibrosis: a bronchoscopic study. Pulmozyme Pediatric Broncoscopy Study Group.

The purpose of this study was to assess the delivery to the lungs and the short-term safety of recombinant human deoxyribonuclease (rhDNase, Pulmozyme) in children with cystic fibrosis younger than 5 years of age compared with older children. Patients between the ages of 3 months and 10 years had bronchoscopic examination with bronchoalveolar lavage (BAL) after administration of an aerosol dose of 2.5 mg of rhDNase. After recovery from the procedure, patients were discharged home for an additional 13 days of rhDNase therapy. During this time adverse events were recorded to assess short-term safety. A total of 98 patients were enrolled, 65 (66%) aged 3 months to 5 years and 33 (34%) aged 5 years to 10 years. Deoxyribonuclease concentrations in BAL fluid were variable (interquartile range, 752 to 3943 micrograms/mL epithelial lining fluid [ELF]) and did not depend on patient age, weight, or height or differ when delivered through a mouthpiece or mask. The median value for the BAL DNA concentration in the younger group was 432 micrograms/mL ELF compared with 703 micrograms/mL ELF in the older patients. This study demonstrates the value of bronchoscopy and BAL for assessing nebulized medication delivery in young children and shows that aerosolized medications can be delivered to and are present in comparable amounts in the lower airways of younger and older children. Exposure to rhDNase appears to be safe over 2 weeks in infants and young children with cystic fibrosis.

Aerosols↗

Evaluation of rat thyroid gland morphophysiological status after three months exposure to 50 Hz electromagnetic field.

Objective of our study was to use morphophysiological criteria in order to determine the sensitivity of male rat thyroid gland to an extremely low frequency electromagnetic field (ELF-EMF) influence and the ability of the gland to repair after period of exposure. Animals were exposed to 50Hz, 50-500 microT ELF-EMF for 3 months when a part of them (group I) were sacrificed, while the rest of animals were subjected to recovery evaluation of the gland and sacrificed after 1 (group II), 2 (group III) and 3 (group IV) weeks. Histological and stereological analyses were performed on paraffin and semifine thyroid gland sections. Serum T3 and T4 were also determined. Histological and stereological analyses showed that the volume density of follicular epithelium and thyroid activation index decreased, while the volume density of colloid and capillary network increased in group I, II and III. The values of all these parameters in group IV were similar to corresponding controls. Serum T3 and T4 concentrations were significantly lower in all exposed animals, except in group I. Results of this study demonstrate that after significant morphophysiological changes caused by ELF-EMF exposure thyroid gland recovered morphologically, but not physiologically, during the investigated repair period.

Animals↗

Systemic glutathione deficiency in symptom-free HIV-seropositive individuals.

To find out whether systemic glutathione deficiency is associated with human immunodeficiency virus (HIV) infection, thus contributing to the immunodeficiency state, glutathione concentrations in venous plasma and lung epithelial lining fluid (ELF) of symptom-free HIV-seropositive and normal individuals were measured. Total and reduced glutathione concentrations in the plasma of the HIV-infected subjects were about 30% of those in the normal individuals. Concentrations of these substances in the ELF of HIV-infected subjects were about 60% of those in the controls. There was no correlation between ELF and plasma concentrations of total or reduced glutathione. Since glutathione enhances immune function, glutathione deficiency may contribute to the progressive immune dysfunction of HIV infection.

Adult↗