We investigated the effects on human keratinocytes (HaCaT) of exposure to a sinusoidal magnetic field of 2 mT (50 Hz). These cells are a good model for studying interaction of nonionising radiation, because they are not shielded from fields in vivo and also because they are resistant to both mechanical and thermal stimuli. We performed scanning microscopy which showed modification in shape and morphology in exposed cells. This modification is related to differential actin distribution as revealed by phalloidin fluorescence analysis. Moreover, the exposed cells show increased clonogenic capacity, as well as increased cellular growth as showed by clonogenicity assays and growth curves. Indirect immunofluorescence analysis using a fluorescent antibody against involucrin and beta4 integrin, which are respectively differentiation and adhesion markers, revealed an increase of involucrin expression and segregation of beta4 integrin in the cell membrane in cells exposed to 50 Hz; a higher percentage of the exposed cells shows a modified pattern of adhesion and differentiation markers. We also present evidence that exposure of HaCaT cells can interfere with protein kinase activity. Our observations confirm the hypothesis that electromagnetic fields at 50 Hz may modify cell membrane morphology and interfere with initiation of the signal cascade pathway and cellular adhesion.
Key words. Atomic force microscopy, β 4 integrin, extremely low frequency, scanning near-field optical microscopy.
SummaryIn this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker β 4 integrin, which revealed an increase of β 4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.
Membrane receptors for rubella virus (RV) in Vero cells were studied by means of two different approaches: (i) by enzyme treatment of the whole cell membrane and (ii) by testing the ability of isolated plasma membrane molecules to compete with cells for virus binding. The replication of RV was studied with both indirect immunofluorescence assay and molecular hybridization techniques. Phospholipases A2 and C digestion of cells greatly reduced the infectivity by the virus, pointing towards the involvement of lipid structures as receptor sites for RV. Furthermore, susceptibility of Vero cells to virus infection was also reduced after beta-N-acetyl-D-glucosaminidase, alpha-glucosidase and beta-galactosidase treatment, suggesting that carbohydrate residues may participate in a complex cellular receptor structure for RV. When the major membrane lipids were examined separately for their ability to inhibit viral infectivity, several phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin) and glycolipids (gangliosides, lactosylceramide, cerebroside sulphate) showed a strong neutralizing activity, confirming the role of membrane lipid moiety in the cell surface receptor for RV.
The wish of this work is the study of the effect of electromagnetic (EMF) radiations at a frequency of 50 Hz on the development of cerebellar granule neurons (CGN). Granule neurons, prepared from newborn rat cerebellum (8 days after birth), were cultured after plate-seeding in the presence of EMF radiations, with the plan of characterizing their cellular and molecular biochemistry, after exposure to the electromagnetic stimulus. Five days challenge to EMF radiations showed, by the cytotoxic glutamate (Glu) pulse test, a 30% decrease of cells survival, while only 5% of mortality was reported for unexposed sample. Moreover, blocking the glutamate receptor (GluR) with the Glu competitor MK-801, no toxicity effect after CGN challenge to EMF radiations and Glu was detected. By patch-clamp recording technique, the Kainate-induced currents from 6 days old exposed CGN exhibited a significant increase with respect to control cells. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses show that EMF exposure of rats CGN, induces a change in both GluRs proteins and mRNAs expression with respect to control. In addition, the use of monoclonal antibody raised against neurofilament protein (NF-200) reveals an increase in NF-200 synthesis in the exposed CGN. All these results indicate that exposure to non-ionizing radiations contribute to a premature expression of GluRs reducing the life span of CGN, leading to a more rapid cell maturation.
The natural and semisynthetic carbohydrates scleroglucan, locust bean gum, tamarind gum (glyloid) and its three sulphate derivatives (GP4311, GP4327 and GP4324), glycogen and its two sulphate derivatives (GP4427 and GP4435), alginic acid and dextran sulphate, were investigated for their inhibitory effect on rubella virus (RV) infection of Vero cells. The neutral polymer scleroglucan and two highly negatively charged compounds, glyloid sulphate 4324 and dextran sulphate, had the highest inhibitory effect on RV antigen synthesis. The antiviral properties of active molecules appears to be dependent on the shape of the macromolecule and/or on the electric charge, while saccharide units play a minor role. The results indicated that polysaccharides blocked a step in virus replication subsequent to virus attachment, such as internalization and/or uncoating. Confirmation that the inhibitory activity of the compounds was directed at the early steps of RV multiplication, was that none of the polysaccharides had any effect on infection initiated by transfection of cells with RVRNA.
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