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Biomedical subjects

E Walum

Publications and source records attributed to E Walum.

At least 19 recordsLinked to original sources
PubMed2003

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery.

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

Acrylamide↗
PubMed2001

Insulin-like growth factor type 1 upregulates uncoupling protein 3.

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.

Carrier Proteins↗
PubMed2000

Expression and intracellular localization of leptin receptor long isoform-GFP chimera.

The leptin receptor (OBR) and its ligand leptin (OB) are key players in the regulation of body weight. The OBR is a member of the class I cytokine receptor family and is alternatively spliced into at least six different isoforms. The multiple forms are identical in their extracellular and transmembrane regions but differ in lengths. The two predominant isoforms include a long form (OBR(l)) with an intracellular domain of 303 amino acids and a shorter form (OBR(s)) with an intracellular domain of 34 amino acids. We have constructed a recombinant OBR(l) chimera with the green fluorescent protein (GFP) by fusing GFP to the C-terminus of the OBR(l). The OBR(l)-GFP chimera was transiently transfected and expressed in SHSY5Y and HEK293 cells. In a STAT-Luciferase assay we show that the GFP moiety in this chimera did not affect the signalling capacity of OBR(l)-GFP. In both SHSY5Y and HEK293 cells transfected with OBR(l)-GFP, a predominant intracellular green OBR(l)-GFP fluorescence was detected in vesicles also positive for internalized fluorophore conjugated leptin. We also found that treatment with the lysosomotropic reagent monensin did not relocalize OBR(l)-GFP together with the human transferrin receptor in recycling endosomes, indicating OBR(l)-GFP not to participate in this pathway. In biotinylation-streptavidin pulse chase experiments, using antibodies raised against GFP and OBR, we observed that the rate of early appearance of OBR(s) at the cell surface, upon leptin stimulation, was faster than that found for OBR(l)-GFP. Taken together, our results provide novel data concerning the intracellular trafficking of the two different isoforms of the leptin receptor.

Biotinylation↗
PubMed2000

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk-1, Flt-1 and Neuropilin-1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase-2 (COX-2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2alpha (PGF2alpha) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2alpha and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Animals↗
PubMed2000

Characterization of leptin intracellular trafficking.

Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR) is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBR) with an intracellular domain of 303 amino acids and a shorter form (OBR) with an intracellular domain of 34 amino acids. Since OBR, is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3-transferrin containing compartments at 37 degrees C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF) and the lysosomal marker protein lamp-1. The transport of leptin was also shown to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.

Anti-Bacterial Agents↗
PubMed1998

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): validation and acute toxicity testing.

Scientific principles demand that before newly developed alternative methods for safety testing are fully embraced by the industrial or regulatory community, they reliably and reproducibly predict the designated toxic end point. The process used to determine reliability and reproducibility is termed validation, and it generally culminates with a highly controlled, blinded study using multiple chemicals and laboratories. It is imperative that the validation study is designed to confirm the previously established reproducibility and predictive power of the assay. Much has been learned recently about the practical aspects of validation through investigation of alternative methods for acute toxicity testing, i.e., those methods that assess acute systemic toxicity, skin irritation, and eye irritation. Although considerable progress has been made--many alternative tests are now commonly used in various industrial settings--there have been few tests that have successfully passed a complete validation. Some of the barriers to successful validation have been a) lack of high-quality, reproducible animal data; b) insufficient knowledge of the fundamental biologic processes involved in acute toxicity; and c) the development of truly robust in vitro assays that can accurately respond to materials with a wide range of chemical and physical characteristics. It is recommended that to progress in the areas of eye and skin irritation we need to expand our knowledge of toxic markers in humans and the biochemical basis of irritation; progress in the area of acute systemic toxicity will require the development of in vitro models to determine gastrointestinal uptake, blood-brain barrier passage, and biotransformation.

Animal Testing Alternatives↗
PubMed1998

Acute oral toxicity.

The purposes of acute toxicity testing are to obtain information on the biologic activity of a chemical and gain insight into its mechanism of action. The information on acute systemic toxicity generated by the test is used in hazard identification and risk management in the context of production, handling, and use of chemicals. The LD50 value, defined as the statistically derived dose that, when administered in an acute toxicity test, is expected to cause death in 50% of the treated animals in a given period, is currently the basis for toxicologic classification of chemicals. For a classical LD50 study, laboratory mice and rats are the species typically selected. Often both sexes must be used for regulatory purposes. When oral administration is combined with parenteral, information on the bioavailability of the tested compound is obtained. The result of the extensive discussions on the significance of the LD50 value and the concomitant development of alternative procedures is that authorities today do not usually demand classical LD50 tests involving a large number of animals. The limit test, the fixed-dose procedure, the toxic class method, and the up-and-down methods all represent simplified alternatives using only a few animals. Efforts have also been made to develop in vitro systems; e.g., it has been suggested that acute systemic toxicity can be broken down into a number of biokinetic, cellular, and molecular elements, each of which can be identified and quantified in appropriate models. The various elements may then be used in different combinations to model large numbers of toxic events to predict hazard and classify compounds.

Administration, Oral↗
PubMed1996

Polygodial induces inositol phosphate turnover in human neuroblastoma SH-SY5Y cells.

The pungent sesquiterpenoid unsaturated dialdehydes polygodial and isovelleral, have previously been shown to increase the intracellular free calcium concentration [Ca2+]i in human neuroblastoma SH-SY5Y cells, partly by a release from intracellular Ca2+ stores, whereas the non-pungent compound epipolygodial, had no effect on the [Ca2+]i. In this study, we investigated the effect of isovelleral, polygodial and epipolygodial on inositol phosphate (IP) formation on the assumption that there might be a correlation between the release of intracellular Ca2+ and pungency of the compounds. It was found that polygodial induced IP mobilization in a concentration dependent way, whereas isovelleral had no effect on the IP formation in the SH-SY5Y cells. Phosphoinositide (PPI) turnover was activated by epipolygodial, but only at concentrations 40-fold higher than for polygodial, which emphasizes the importance of the correct stereometry in the dialdehyde configuration for the biological activity of polygodial. The polygodial-induced formation of IP1 was reduced by 71% under extracellular calcium-free conditions, which suggests feedback interactions between the IP formation and the increase in [Ca2+]i to account for a periodic activation of phospholipase C(PLC).

Calcium↗
PubMed1995

Sodium-dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat.

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 microM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate alpha-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na+, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na+ by Li+ in the perfusion buffer. We conclude that the co-transport of Na+, in the Na(+)-dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na+/K(+)-ATPases.

Animals↗
PubMed1995

Use of primary cultures and continuous cell lines to study effects on astrocytic regulatory functions.

1. Current opinions on the mechanisms for glutamate-mediated neurotoxicity are reviewed. The protective role of astrocytic high-affinity glutamate transport is also discussed. 2. Low-density seeding of primary astrocytes from rat hemispheres was found to result in the development of reactive-like astrocytes. Typical glial signs of amyotrophic lateral sclerosis (ALS) could not be induced in astrocyte cultures by serum from ALS-patients. 3. Glutamate (100 mumol/L) was found to induce an increase in respiratory activity in primary cultures of astrocytes. This stimulation appeared to be related to the co-transport of Na2+ with glutamate and a resulting activation of Na2+/K(+)-ATPase. Both basal respiration and glutamate-stimulated oxygen consumption was inhibited by organic solvents. 4. Preliminary results show that heavy metals cause an increase in the mitochondrial DNA content at concentrations that have no effect on growth rate or morphology in a glial cell line. This increase was accompanied by an inhibition of oxygen consumption and an increased production of lactate at unaltered ATP levels.

Amyotrophic Lateral Sclerosis↗
PubMed1994

Sesquiterpenoid unsaturated dialdehydes increase the concentration of intracellular free Ca2+ in human neuroblastoma SH-SY5Y cells.

The effect of the three sesquiterpenoid unsaturated dialdehydes--polygodial, isovelleral, and epipolygodial--on intracellular free Ca2+ concentration, [Ca2+]i, was investigated in the human neuroblastoma cell line SH-SY5Y. [Ca2+]i was measured by the Fura-2 spectrophotofluorometric technique in multi-cell and single-cell experiments. Polygodial and isovelleral induced an increase in [Ca2+]i at low concentrations (0.1 and 0.5 micrograms/ml, respectively) but epipolygodial affected [Ca2+]i only at a high concentration (10 micrograms/ml). The results indicated that there was a relationship between the effect on [Ca2+]i and the pungency of the sesquiterpenes tested. Experiments in a Ca(2+)-free extracellular solution showed that Ca2+ was also released from intracellular calcium stores. Images from single-cell experiments indicated that polygodial induced fluctuations in the [Ca2+]i in some cells. The mechanism behind the sesquiterpene induced increase of the [Ca2+]i was not identified but a possible mobilization of inositol phosphates is considered.

Aldehydes↗
PubMed1994

Reactive gliosis and monoamine oxidase B.

A double-staining method was applied to cryosections of human spinal cord from patients who died with amyotrophic lateral sclerosis (ALS) and corresponding controls in order to investigate cellular content of monoamine oxidase B (MAO-B). 3H-L-Deprenyl emulsion autoradiography was used in combination with histochemical methods for the detection of astrocytes and monocytes/microglia. In the ALS spinal cords an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes was demonstrated. No significant 3H-L-deprenyl binding was observed in cells derived from the mesoderm, e.g. monocytes or microglia. Furthermore, a sub-population of reactive astrocytes that contained low levels of MAO-B was observed in spinal sections. These findings were further substantiated by studies performed on primary astrocyte cultures.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Monoamine oxidase-B in astrocytes.

In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.

Amyotrophic Lateral Sclerosis↗