The labile iron pool: characterization,measurement, and participation in cellular processes(1)

The cellular labile iron pool (LIP) is a pool of chelatable and redox-active iron, which is transitory and serves as a crossroad of cell iron metabolism. Various attempts have been made to analyze the levels of LIP following cell disruption. The chemical identity of this pool has remained poorly characterized due to the multiplicity of iron ligands present in cells. However, the levels of LIP recently have been assessed with novel nondisruptive techniques that rely on the application of fluorescent metalosensors. Methodologically, a fluorescent chelator loaded into living cells binds to components of the LIP and undergoes stoichiometric fluorescence quenching. The latter is revealed and quantified in situ by addition of strong permeating iron chelators. Depending on the intracellular distribution of the sensing and chelating probes, LIP can be differentially traced in subcellular structures, allowing the dynamic assessment of its levels and roles in specific cell compartments. The labile nature of LIP was also revealed by its capacity to promote formation of reactive oxygen species (ROS), whether from endogenous or exogenous redox-active sources. LIP and ROS levels were shown to follow similar "rise and fall" patterns as a result of changes in iron import vs. iron chelation or ferritin (FT) degradation vs. ferritin synthesis. Those patterns conform with the accepted role of LIP as a self-regulatory pool that is sensed by cytosolic iron regulatory proteins (IRPs) and feedback regulated by IRP-dependent expression of iron import and storage machineries. However, LIP can also be modulated by biochemical mechanisms that override the IRP regulatory loops and, thereby, contribute to basic cellular functions. This review deals with novel methodologies for assessing cellular LIP and with recent studies in which changes in LIP and ROS levels played a determining role in cellular processes.     

Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.     

Nonionic, Water Self-Dispersible "Hairy-Rod" Poly(p-phenylene)-g-poly(ethylene glycol) Copolymer/Carbon Nanotube Conjugates for Targeted Cell Imaging

The generation and fabrication of nanoscopic structures are of critical technological importance for future implementations in areas such as nanodevices and nanotechnology, biosensing, bioimaging, cancer targeting, and drug delivery. Applications of carbon nanotubes (CNTs) in biological fields have been impeded by the incapability of their visualization using conventional methods. Therefore, fluorescence labeling of CNTs with various probes under physiological conditions has become a significant issue for their utilization in biological processes. Herein, we demonstrate a facile and additional fluorophore-free approach for cancer cell-imaging and diagnosis by combining multiwalled CNTs with a well-known conjugated polymer, namely, poly(p-phenylene) (PP). In this approach, PP decorated with poly(ethylene glycol) (PEG) was noncovalently (π-π stacking) linked to acid-treated CNTs. The obtained water self-dispersible, stable, and biocompatible f-CNT/PP-g-PEG conjugates were then bioconjugated to estrogen-specific antibody (anti-ER) via -COOH functionalities present on the side-walls of CNTs. The resulting conjugates were used as an efficient fluorescent probe for targeted imaging of estrogen receptor overexpressed cancer cells, such as MCF-7. In vitro studies and fluorescence microscopy data show that these conjugates can specifically bind to MCF-7 cells with high efficiency. The represented results imply that CNT-based materials could easily be fabricated by the described approach and used as an efficient "fluorescent probe" for targeting and imaging, thereby providing many new possibilities for various applications in biomedical sensing and diagnosis.     

Lipid peroxidation of a human hepatoma cell line (HepG2) after incorporation of linoleic acid, arachidonic acid, and docosahexaenoic acid

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography-mass spectrometry (GC-MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H(2)O(2) (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H(2)O(2). However, the enhancement of lipid peroxidation by H(2)O(2) was not observed in DHA-supplemented cells. The same result was obtained in the GC-MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA-MHP and a small amount of DHA-MHP were observed in AA- and DHA-supplemented cells respectively. GC-MS analysis also indicated the specific positional distribution of DHA-MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.     

The "side-effects" of plasmids (R-plasmids and virulence)

Many plasmids affect the host cells. Their effects cannot be explained only by the expression of the well-known genes coding for antibioticresistance, bacteriocinogeny and hemolysis or the analogous genes (side-effects). The side effects are not characteristic of all plasmids operating under similar conditions. Forecasting of the side-effects inducikility by any definite plasmid is impossible now. Sometimes the same functions exert the contrary effects on the bacterial cell. The connection between the presence of plasmids, especially R-plasmids and the complex cellular property, virulence, is of great interest. Often, bacteria become less virulent obtaining the plasmids. Two possible reasons causing such an effect are discussed. The first one is a direct effect of plasmids on cellular physiology. The second reason is connected with population shifts caused by the fact that the cells with initial low virulence possess the recipient ability predominantly. The decreased virulence of bacteria harbouring R-plasmids, in authors opinion, is quite a natural phenomenon based on plasmid host cells adaptation to the existence in "the realm of antimicrobial agents".     

Reprint of "Improved spectral resolution in COSY (1)H NMR spectra of proteins via double quantum filtering"

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. It has been shown that expanded polyQ tract-containing proteins impair the functions of other cellular proteins. However, quantitative changes of cellular proteins in cells expressing expanded polyQ tract-containing proteins have not been performed. Here, we performed proteomic analysis of cells expressing expanded polyQ tract-containing proteins, and showed that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24). In addition, we revealed that down-regulation of GRP78 expression resulted in increase of the aggregation of EGFP-polyQ97. Conversely, the aggregation of EGFP-polyQ97 was suppressed by the overexpression of GRP78 in the cells. Furthermore, it seemed that the decreased GRP78 expression in the cells expressing EGFP-polyQ97 was due to the enhanced protein degradation of GRP78 through the ubiquitin-proteasome pathway. These findings indicated that GRP78, which has an inhibitory effect on the aggregation of proteins containing expanded polyQ tract, may be an effective target for the treatment of polyQ diseases.     

Absorption and paracellular visualization of fluorescein, a hydrosoluble probe, in intact house sparrows (Passer domesticus)

We describe a method to visualize the cellular location of compounds during absorption by the small intestine in intact animals. First, we employed pharmacokinetic methodology to measure the fractional absorption of sodium fluorescein, a small (MW = 376) water-soluble molecule that is widely used as hydrophilic marker molecule for paracellular permeability studies. Based on the hypothesis that the paracellular pathway acts as a sieve, we predicted that fluorescein absorption would be considerable, but less than that of passively absorbed L-glucose which is a smaller molecule (MW = 180). When the two compounds were gavaged into house sparrows simultaneously, the birds absorbed significantly less fluorescein (42 +/- 8%) than L-glucose (82 +/- 7%), as predicted, and absorptions of the two were correlated as one would predict if they shared the same pathway. We removed intestinal tissue 10 min after gavage with sodium fluorescein and determined the cellular location of the compound's fluorescence using confocal laser microscopy. The fluorescent signal was found primarily in the paracellular space. In contrast, in the same type of experiment using instead the similar-sized fluorescent lipophilic compound rhodamine 123 (MW = 381), most fluorescence appeared inside enterocytes, as expected for a compound that diffuses across the apical membrane. Thus, results from all the experiments are consistent with the hypothesis that hydrophilic fluorescein is absorbed primarily via a paracellular pathway. These methods could be applied to visualize absorption pathways of other compounds in other intact animals.     

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.     

A "ready-to-use" fluorescent-labelled-cysteine-TBTP (4-thiobutyltriphenylphosphonium) synthon to investigate the delivery of non-permeable PNA (peptide nucleic acids)-based compounds to cells

This paper reports: (i) the facile synthesis of a cysteine synthon incorporating both a fluorescent group and a triphenylphosphonium derivative (TBTP) via the formation of a disulphide bond, which can subsequently undergo facile intracellular scission, (ii) the direct conjugation of this synthon to a non-permeable drug, (a cyclic PNA (peptide nucleic acid)-based compound has been chosen as a model), and (iii) that this conjugation enables the efficient homogenous delivery of the otherwise non-permeable cyclic PNA into the cytoplasm of cells, as demonstrated by fluorescence microscopy. Our results indicate that this fluorescent-labelled cysteine-TBTP synthon can provide a very useful tool for exploring the cellular uptake of a large range of molecules of biological interest, containing only a single reactive function. The preparation of an activated TBTP derivative is also described and this procedure could be widely used to introduce a TBTP cation to any thio-containing molecule.     

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.     

Measles virus-induced disruption of the glial-fibrillary-acidic protein cytoskeleton in an astrocytoma cell line (U-251)

A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been used to infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton. Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal components. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells. No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type, whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells were quantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another intermediate filament on which MVeGFP has no effect, as a control.     

Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS

BACKGROUND: E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS: High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS: The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.     

Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism

Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP(-*) anion. Prolonged continuous wave illumination at the peak of YFP(-) absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP(-) in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP(-) within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Dual-rhodamine urea derivative, a novel chemidosimeter for Hg(II) and its application in imaging Hg(II) in living cells

Abstract  The synthesis and spectral properties of a chemidosimeter 1,4-di[2-(6-ethylamino-3-ethylimino-2,7-dimethyl-3H-xanthen-9-yl) benzoic acid (aminomethyl)-3-phenylthiourea] benzene (1) for Hg(II) ions are reported, and it has been demonstrated that 1 can be used as a fluorescent probe for monitoring Hg(II) ions in living cells. Graphical abstract  A highly sensitive fluorescent probe (1) was developed as a fluorescent and colorimetric chemodosimeter in dimethyl sulfoxide/methanol solution with a broad pH range (pH 5–10) and high selectivity toward Hg²⁺ ions but no significant response toward other competitive cations. Furthermore, by means of confocal laser scanning microscopy experiments, it is demonstrated that 1 can be used as a fluorescent probe for monitoring Hg²⁺ in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26)

Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.     

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.     

Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas. Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin-stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis. Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.     

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK)

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.