Effects of Magnetic Field on Phenol Biodegradation and Cell Physiochemical Properties of Rhodococcus erythropolis

Using phenol-degrading Rhodococcus erythropolis cells, the stimulative effect of a homogenous electromagnetic field (EMF) (magnetic induction 10–130 mT) on the growth and utilization of phenol (0.3–1.2 g/L) was investigated. Similarly, the EMF effect was tested on a R. erythropolis biofilm formation, which was found to increase the cell adhesion abilities significantly. Detected magnetic stimulation of cell adhesion disposition was supplemented with the results of cell surface hydrophobicity and chemical composition analysis.     

Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix

The fibroblast cell line Balb/c 3T3 makes both close and tight-focal adhesive contacts with the plasma fibronectin (pFn)-coated tissue culture substratum. Detachment of these cells mediated by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid leaves tight-focal contacts and a subset of close contacts as substratum-attached material (SAM). The enrichment of heparan sulfate (HS) proteoglycan (HS-PG) in SAM under specific attachment conditions, as well as the recent demonstration of HS binding to pFn or cellular Fn, has evolved a series of experiments with the selective HS-binding protein platelet factor-4 to analyze the requirement of the HS-binding activity of pFn in the formation of these two types of adhesive contacts. In addition, the cell-binding domain (CBD) of pFn, which recognizes an unidentified cell surface receptor, has been isolated free of HS-binding domains after proteolytic cleavage of pFn. These functional studies indicate that the binding of pFn on the substratum to cell surface HS-PG is necessary and sufficient to generate close contacts with transmembrane signaling of this proteoglycan to reorganize lengthy microfilament bundles in the cytoplasm. Cells spread incompletely on CBD alone, form only close contacts, and reorganize highly concentrated actin filaments only in their spiky projections. Furthermore, the formation of tight-focal contacts with associated stress fibers appears to require both reactions of pFn, binding to cell surface HS-PG and to its unidentified receptor. The importance of HS-PG in these functional studies has led to its better biochemical characterization in SAM. Initially formed close contacts of these cells contain principally a large HS-PG that aggregates into high-molecular-weight complexes by some unknown mechanism. With time, there are two different mechanisms of catabolism of HS-PG, one of which includes the liberation of single-chain HS. The importance of these changes in the HS-PG in SAM are now being analyzed with regard to the formation and disappearance of close and tight-focal adhesions.     

Therapeutic potential of electromagnetic fields for tissue engineering and wound healing

Ability of electromagnetic fields (EMF) to stimulate cell proliferation and differentiation has attracted the attention of many laboratories specialized in regenerative medicine over the past number of decades. Recent studies have shed light on bio‐effects induced by the EMF and how they might be harnessed to help control tissue regeneration and wound healing. Number of recent reports suggests that EMF has a positive impact at different stages of healing. Processes impacted by EMF include, but are not limited to, cell migration and proliferation, expression of growth factors, nitric oxide signalling, cytokine modulation, and more. These effects have been detected even during application of low frequencies (range: 30–300 kHz) and extremely low frequencies (range: 3–30 Hz). In this regard, special emphasis of this review is the applications of extremely low‐frequency EMFs due to their bio‐safety and therapeutic efficacy. The article also discusses combinatorial effect of EMF and mesenchymal stem cells for treatment of neurodegenerative diseases and bone tissue engineering. In addition, we discuss future perspectives of application of EMF for tissue engineering and use of metal nanoparticles activated by EMF for drug delivery and wound dressing.     

Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1–1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects. Bioelectromagnetics 19:85–91, 1998. © 1998 Wiley-Liss, Inc.     

Can prenatal exposure to a 900 MHz electromagnetic field affect the morphology of the spleen and thymus,and alter biomarkers of oxidative damage in 21-day-old male rats?

We investigated the effects of a 900 Megahertz (MHz) electromagnetic field (EMF), applied during the prenatal period, on the spleen and thymus of 21-day-old male rat pups. Pregnant Sprague-Dawley rats were divided into control and EMF groups. We applied 900 MHz EMF for 1 h/day to the EMF group of pregnant rats. Newborn male rat pups were removed from their mothers and sacrificed on postnatal day 21. Spleen and thymus tissues were excised and examined. Compared to the control group, thymus tissue malondialdehyde levels were significantly higher in the group exposed to EMF, while glutathione levels were significantly decreased. Increased malondialdehyde and glutathione levels were observed in splenic tissue of rats exposed to EMF, while a significant decrease occurred in superoxide dismutase values compared to controls. Transmission electron microscopy showed pathological changes in cell morphology in the thymic and splenic tissues of newborn rats exposed to EMF. Exposure to 900 MHz EMF during the prenatal period can cause pathological and biochemical changes that may compromise the development of the male rat thymus and spleen.     

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

T cell receptor binding transition states and recognition of peptide/MHC

T cell receptor recognition of peptide/MHC has been described as proceeding through a "two-step" process in which the TCR first contacts the MHC molecule prior to formation of the binding transition state using the germline-encoded CDR1 and CDR2 loops. The receptor then contacts the peptide using the hypervariable CDR3 loops as the transition state decays to the bound state. The model subdivides TCR binding into peptide-independent and peptide-dependent steps, demarcated at the binding transition state. Investigating the two-step model, here we show that two TCRs that recognize the same peptide/MHC bury very similar amounts of solvent-accessible surface area in their transition states. However, 1300-1500 A2 of surface area is buried in each, a significant amount suggestive of participation of peptide and associated CDR3 surface. Consistent with this interpretation, analysis of peptide and TCR variants indicates that stabilizing contacts to the peptide are formed within both transition states. These data are incompatible with the original two-step model, as are transition state models built using the principle of minimal frustration commonly employed in the investigation of protein folding and binding transition states. These findings will be useful in further explorations of the nature of TCR binding transition states, as well as ongoing efforts to understand the mechanisms by which T cell receptors recognize the composite peptide/MHC surface.     

The formation of synapses in the central nervous system

Interneuronal synapses are specialized contact zones formed between the transmitting pole of one neuron, usually an axon, and the receptive pole of another nerve cell, usually a dendritic process or the soma. The formation of these synaptic contacts is the result of cellular events related to neurite elongation, the establishment of polarity, axon guidance, and target recognition. A series of morphological rearrangements takes place once synaptic targets establish their initial contact. These changes include the clustering of synaptic vesicles in the presynaptic element and the formation of a specialized area capable of signal transduction at the postsynaptic target. The present review discusses the role of different synaptic proteins in the cellular events leading to the formation of synapses among neurons in the central nervous system.     

Dynamic study of the transition from hyaluronan- to integrin-mediated adhesion in chondrocytes

Membrane-bound hyaluronan mediates the initial adhesive interactions between many cell types and external surfaces. In RCJ-P chondrocytes, such early contacts are mediated through a thick hyaluronidase-sensitive coat. The early adhesion is followed by integrin-mediated interactions and the formation of stable focal adhesions. During this process, the distance between the cell membrane and the surface is reduced from micrometers to few tens of nanometers. The transition from hyaluronan- to integrin-mediated adhesion was studied on glass surfaces by total internal reflection fluorescence microscopy. Hyaluronan-mediated adhesion precedes focal adhesions formation by 2-10 min. After these initial interactions, the pericellular hyaluronan remains sequestered into discrete pockets between the cell and the surface, which are a few hundreds nanometers thick and a few micrometers wide, and are flanked by focal adhesions. The hyaluronan coat facilitates the nucleation of small paxillin-rich contacts, which later mature into focal adhesions. These dynamic studies demonstrate that pericellular hyaluronan mediates initial cell-surface adhesion, and regulates the formation of focal adhesions.     

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G₀/G₁ phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15–60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.     

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.     

Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study

To investigate the effects of low frequency electromagnetic fields (EMF) on the proliferation of epidermal stem cells, human epidermal stem cells (hESC) were isolated, expanded ex vivo, and then exposed to a low frequency EMF. The test and control cells were placed under the same environment. The test cells were exposed for 30 min/day to a 5 mT low frequency EMF at 1, 10, and 50 Hz for 3, 5, or 7 days. The effects of low frequency EMF on cell proliferation, cell cycle, and cell‐surface antigen phenotype were investigated. Low frequency EMF significantly enhanced the proliferation of hESC in the culture medium in a frequency‐dependent manner, with the highest cell proliferation rate at 50 Hz (P < 0.05). Exposure to a low frequency EMF significantly increased the percentage of cells at the S phase of the cell cycle, coupled with a decrease in the percentage of cells in the G1 phase (P < 0.05) but the effect was not frequency dependent. The percentage of CD29⁺/CD71^? cells remained unchanged in the low frequency EMF‐exposed hESC. The results suggested that low frequency EMF influenced hESC proliferation in vitro, and this effect was related to the increased proportion of cells at the S phase. Bioelectromagnetics 34:74–80, 2013. © 2012 Wiley Periodicals, Inc.     

The normalization of tumor cell morphology related to an increase in their size: research on giant cells produced by mitomycin C treatment]

This study shows that artificial increase in cell site leads to morphological normalization of transformed fibroblasts. Mouse L cells (clone 171/5) were used. As most transformed cells, they were poorly spread on the substratum, made only dot-like focal contacts with it, rounded quickly at room temperature and did not contain prominent actin cables. Giant cells were obtained by incubation of these cells in the medium supplemented with mitomycin C (0.15-0.20 mcg/ml). DNA synthesis and mitosis were blocked by this treatment, while protein synthesis was changing very slightly. As a consequence, the cell size increased dramatically from 3 to 11 days of the cell incubation in the mitomycin containing medium. The degree of cell spreading per mcg of protein increased significantly in the giant cells. These cells do not round after moderate cooling, and well developed system of actin cables and matured streak-like focal contacts associated with these cables are formed in them. These results, along with our previous data on the restoration of cell spreading and cytoskeleton structure in giant multinucleated cells, provide strong evidences that the increase in cell size per se can induce qualitative changes in cell morphology. It can be suggested that there are some scaling-dependent factors regulating the processes of cytoskeleton assembly and formation of cell-substrate contacts.     

The dynamics and mechanics of endothelial cell spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.     

In vitro exposure to electromagnetic fields: changes in tumour cell properties

Two human colon cancer cell lines, Colo 205 and Colo 320 DM, have been studied for their responses to 60 Hz-generated electromagnetic fields (EMF) using soft agar cloning and monoclonal antibody binding assays to assess exposure-induced changes. Cellular responses have been studied after 24 h continuous exposure of cells concurrently to four experimental conditions; i.e. no EMF (E-M-), magnetic field only (M+, 1.0 G rms), electric field only (E+, 300 mA/m2 rms), and combined electric plus magnetic fields at these intensities (E+M+). Under these conditions, both cell lines demonstrated significantly increased colony formation in soft agar and increased expression of tumor associated antigens after exposure to E+M+ and to M+ as compared to unexposed controls.     

Substratum contacts and cytoskeletal reorganization of BALB/c 3T3 cells on a cell-binding fragment and heparin-binding fragments of plasma fibronectin

BALB/c 3T3 cells make both close contacts and tight-focal contacts (with associated microfilament stress fibers) on plasma fibronectin (pFN)-coated substrata. To resolve the importance of the heparan sulfate-binding or cell-binding activities of the pFN molecule in these adhesive responses, a cell-binding fragment (120K) (CBF) free of any heparan sulfate-binding activity was prepared from human pFN by chymotrypic digestion and isolated as described by Pierschbacher et al. (Cell 26 (1981) 259). These adhesive responses to CBF were also compared to those of the model heparan sulfate-binding protein, platelet factor-4 (PF4), or heparin-binding fragments (HBF) of pFN. On intact pFN, greater than 70% of the cells formed tight-focal contacts and associated stress fibers by 4 h, the latter staining with NBD-phallacidin. In contrast, cells spread differently on CBF and failed to form tight-focal contacts; staining with NBD-phallacidin was localized to spiky projections at the cell margin with no detectable stress fiber formation. On PF4 or HBF, cells failed to form tight-focal contacts but did spread well and formed long microfilament bundles in peripheral lamellae. Spreading on CBF, HBF, or PF4 was paralleled by formation of close contacts. Spreading and to some extent attachment of cells on CBF was inhibited with a small peptide containing the Arg-Gly-Asp-Ser sequence; responses on HBF were unaffected by this peptide. When mixtures of CBF and PF4 were tested, cells still failed to form tight-focal contacts and stress fibers. These results demonstrate that the binding of CBF to its probable receptor under conditions routinely used to assay spreading activity results in an incomplete adhesive response compared with intact pFN. While this partial response may result from quantitative differences in the density of active cell-binding domains on the substratum, the pattern of microfilament reorganization produced by the binding of PF4 to cell surface heparan sulfate proteoglycans suggests that the ability of pFN to promote formation of tight-focal contacts and stress fibers may reside in the coordinate interaction of two or more binding activities in the intact molecule.     

Crystal packing analysis of Rhodopsin crystals

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin-rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.     

ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin

ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the beta1-integrin. Furthermore, ADAM15 accumulation in cell-cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15-GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15-GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.