[Effect of 33 Hz Pulsed Electric Field on Insulin Amyloidosis].

The destabilizing of protein leads to self-aggregation and fibrillar assemblies. In the form of amyloid fibrils or fibril precursors, protein not only lacks the original biological function but also may be harmful to organisms. Stimulated by an intense electric field, the secondary structures of protein can be disturbed and transfer to aggregations or unfolding conformations, which may inhibit the fibrillation process. As a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. This project is focusing on in-vitro studies employing a 33 Hz pulsed electric field (PEF) to see if there is possible causal connection between insulin fibrillation and PEF exposure. Thioflavin T (ThT)-fluorescence, circular dichroism(CD) and transmission electron microscopy (TEM) techniques were employed regarding the effects of exposure duration and field intensity of the PEF on the fibrillation mechanism of insulin. The results confirm that the PEF exposed insulin molecules may primitively have a slight change in its native structure, causing aggregation. The aggregates in the PEF exposed insulin solution are difficult to dissolve to facilitate the unfolding of insulin molecules. When the molecular conformation converts from α-helical to ß-sheet structure, the fibrillation velocity in the PEF exposed insulin is accelerated by the PEF exposure thereby shortening the lifetime of the intermediates. The morphology of mature fibrils changes from long twisted fibrils to shorter and less matured fibrils. All these effects enhance when the exposure duration and electric intensity increase. The investigated evidences suggest that the PEF can inhibit insulin amyloidosis.

Weak power frequency magnetic field acting similarly to EGF stimulation, induces acute activations of the EGFR sensitive actin cytoskeleton motility in human amniotic cells.

In this article, we have examined the motility-related effects of weak power frequency magnetic fields (MFs) on the epidermal growth factor receptor (EGFR)-sensitive motility mechanism, including the F-actin cytoskeleton, growth of invasive protrusions and the levels of signal molecules in human amniotic epithelial (FL) cells. Without extracellular EGF stimulation, the field stimulated a large growth of new protrusions, especially filopodia and lamellipodia, an increased population of vinculin-associated focal adhesions. And, an obvious reduction of stress fiber content in cell centers was found, corresponding to larger cell surface areas and decreased efficiency of actin assembly of FL cells in vitro, which was associated with a decrease in overall F-actin content and special distributions. These effects were also associated with changes in protein content or distribution patterns of the EGFR downstream motility-related signaling molecules. All of these effects are similar to those following epidermal growth factor (EGF) stimulation of the cells and are time dependent. These results suggest that power frequency MF exposure acutely affects the migration/motility-related actin cytoskeleton reorganization that is regulated by the EGFR-cytoskeleton signaling pathway. Therefore, upon the MF exposure, cells are likely altered to be ready to transfer into a state of migration in response to the stimuli.

[Secondary structure changes of insulin induced by PEF exposure at different temperatures using Raman spectra and theoretical model analysis].

Recently, biological effects induced by weak electromagnetic fields have been a public concern. Our previous study found temperature and electromagnetic field co-effects on insulin conformation. Therefore, in the present study, Raman spectroscopy was employed to investigate the secondary structure changes of insulin molecule induced by pulsed electric field (PEF) exposure at various temperatures. The content changes in alpha helix of insulin were obtained. Then, protein helix-random coil transition model was used to quantitatively study the experimental results. The theoretical model could figure out the effect of PEF on alpha helix contents of insulin at different temperatures. The protein secondary structure transits from helix to random coil evoked by PEF exposure and change of thermodynamic environment, which could explain the reason for the decline of alpha helix content of insulin caused by PEF exposure together with temperature rising. The results offer experimental basis and theoretical reference for further study of the mechanism of nonthermal effects of weak electromagnetic fields on biological molecule secondary structure.

[Study on temperature & EMF co-effects on insulin conformation and biological functions by fluorescence and Raman spectroscopy].

Our previous studies had suggested that the intercellular signal molecule might be an important target of electromagnetic fields. Insulin, an intercellule signal molecule, plays a critical role in transferring life information. The studies on effects of pulsed electric fields (PEF) on insulin molecule are meaningful for explaining the mechanism of biological effects of electromagnetic fields. The PEF, which we used, with its highest electric field (2 x 10(6) V x m(-1)) coupled into the insulin buffer, was about 1 V x cm(-1) cm, with a repeating frequency of 50 Hz. In the present study, the changes of insulin conformation induced by PEF were studied by fluorescence spectroscopy. Insulin solution was exposed to 50 Hz PEF with different electric field intensities for 5-35 min, which caused a time-and dose-dependent decrease in fluorescence intensities of insulin. Further, insulin solution was exposed to PEF at different temperatures to investigate the effects of PEF co-operated with temperature on insulin. The results indicated that the difference in temperature (about 5 degrees C) could induce conflict results, which is due to the effects of PEF co-operated with temperature rather than only to the effect of temperature. The authors calculated that the increase in temperature induced by PEF was 0.07 degrees C (less than 0.1 degrees C). So the effects of PEF were scarcely explained by thermal effects, it belongs to "non-thermal effects" of electric fields. So it was concluded that temperature is a considerably important factor in "non-thermal effects" of electric fields, and the ignorance of variety of temperature probably result in the contrary conclusion. Further, Raman spectroscopy was used to investigate the details of structure of insulin treated by PEF co-operated with temperature. The results of Raman spectroscopy verified the effects of PEF co-operated with temperature on insulin. And the reductions of the S-S band intensity at 510 cm(-1), the skeletal C-C stretch band intensity at 934 cm(-1), and the content of the secondary structure of the alpha helix were observed. Both S--S linkages and alpha helix structure were important to the stabilization of insulin conformation. Modification of insulin may change the biological activity either by reducing the affinity of the hormone for the receptor or by decreasing the ability of the complex, when formed, to elicit a biological response.

[Raman spectra of murine skin irradiated by high power millimeter wave].

An experimental study on Raman spectroscopy of normal murine skin and the skin irradiated by high power millimeter wave (HPMM) is presented. It is showed that the Raman spectra of normal skin mainly originate from collagen, and the characteristic peaks are 857, 936 and 1 658 cm(-1). The result showed that after irradiation by HPMM, the relative intensity of the characteristic peaks at 857 and 936 cm(-1) of Raman spectra was decreased. This meant that the collagen was destroyed and even daimaged. It was probably indicated that the skin tissue was damaged and could not be restored. The result also showed that the intensity of the characteristic peak at 1658 cm(-1) of the skin tissue irradiated by millimeter wave with the duration of 20 s decreased. It was considered that the protein in the skin was destroyed. Those results were consistent with macroscopic observation results.

[Effect of 50 Hz power frequency magnetic field on microfilament cytoskeleton assembly of human amnion FL cells].

OBJECTIVE: Investigations were carried out to understand the effect of 50 Hz power frequency magnetic field on microfilament assembly of human amniotic cells and on expression of actin and epidermal growth factor receptor. METHODS: Human amnion FL cells were exposed to 0.1, 0.2, 0.3, 0.4, 0.5 mT power frequency magnetic field for 30 minutes. Microfilaments were marked using Phalloidin-TRITC, and then were observed under a fluorescence microscope. An optical method was used to detect the relative content of microfilament in cells. A scanning electron microscope was used to detect the cell shape. The content of actin and epidermal growth factor receptor in the preparation of the detergent-insoluble cytoskeleton were measured by western-blotting to analyse the potential mechanism of the change induced by magnetic field. RESULTS: Intracellular stress fibers were found to decrease after exposing cells to a 0.2 mT power frequency magnetic field for 30 minutes. New microfilament and filopodia bundles appeared at the cell periphery after exposure, but the detected total F-actin content per cell was not significantly changed, detected by a F-actin-specific dye. The change in the amount of microfilaments caused by the field could be recovered 2 hours later when the field was withdrawn. The mean height of microfilament cytoskeleton decreased from (12.37 +/- 1.28) microm to (9.97 +/- 0.38) microm (t = 6.96, P > 0.05) after exposure using a confocal microscope. The cell shapes became more flat and lamellipodia appeared after exposure observed by a scanning electron microscope. By using Western blotting method, the intracellular contents of epidermal growth factor receptor and of actin in the preparation of the detergent-insoluble cytoskeleton which are associated with high-affinity epidermal growth factor receptors, increased about (31.2 +/- 4.1)% (t = 17.10, P < 0.05) and (16.8 +/- 2.3)% (t = 16.68, P < 0.05) respectively, compared with that of the control. CONCLUSION: These results suggest that a short time exposure to a 0.2 mT power frequency magnetic field induces re-organization of microfilament in human amnion FL cells. These changes could be recovered by field withdraw and may have something with the clustering of epidermal growth factor receptors induced by magnetic field.

[Effects of power frequency magnetic field on Ca2+ transport of skeletal muscle sarcoplasmic reticulum vesicles].

OBJECTIVE: To investigate the effects of power frequency magnetic field on the Ca2+ transport dynamics of isolated sarcoplasmic reticulum vesicles. METHODS: The assays of Ca2+ uptake time course and the Ca2+-ATPase activity of sarcoplasmic reticulum vesicles were investigated by using dynamic mode of spectrometry with a Ca2+ dye; Ca2+ release channel activation was examined by 3H-ryanodine binding and Ca2+ release assays; membrane fluidity of sarcoplasmic reticulum vesicles was examined by fluorescence polarization, without or with exposure to the vesicles at a 0.4 mT, 50 Hz sinusoidal magnetic field. RESULTS: 0.4 mT, 50 Hz sinusoidal magnetic field exposure caused about a 16% decline of the initial Ca2+ uptake rate from a (29.18 +/- 3.90) pmol.mg(-1).s(-1) to a (24.60 +/- 3.81) pmol.mg(-1).s(-1) and a 26% decline of the Ca2+-ATPase activity from (0.93 +/- 0.05) micromol.mg(-1).min(-1) to (0.69 +/- 0.07) micromol.mg(-1).min(-1) of sarcoplasmic reticulum vesicles, whereas caused a 15% increase of the initial Ca2+ release rate from (4.83 +/- 0.82) pmol.mg(-1).s(-1) to (5.65 +/- 0.43) pmol.mg(-1).s(-1) and a 5% increase in 3H-ryanodine binding to the receptor from (1.10 +/- 0.12) pmol/mg to (1.16 +/- 0.13) pmol/mg, respectively. CONCLUSION: The decline of Ca2+-ATPase activity and the increase of Ca2+ release channel activity should result in a down-regulation of Ca2+ dynamic uptake and an up-regulation of Ca2+ release induced by exposing the sarcoplasmic reticulum to a 0.4 mT, 50 Hz power frequency magnetic field.

[Synchronous fluorescence and raman spectroscopy study on the interaction of pulsed electric field (PEF) and bovine serum albumin (BSA)].

The interaction of pulsed electric field (PEF) and bovine serum albumin (BSA) was studied by synchronous fluorescence and Raman spectroscopy. The results of synchronous fluorescence showed that pulsed electric field exerted its effects on the emission fluorescence spectrum and reduced the fluorescence intensities of the tyrosine and tryptophan side chains. The results of Raman spectroscopy verified this. These two experiments indicated that PEF exposure changed the microenvironments of the two aromatic amino adds, which were located in the active parts of BSA, and further indicated the conformational changes of the proteins, and the change in its biological functions.

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis.

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.

[Raman analysis of conformation changes of insulin solvent after being exposed to ELF pulsed electric field].

Raman spectra of insulin solvents are presented before and after being exposed to the pulsed electric field with extremely low frequency 50 Hz (ELF). The covalences of the molecule were not affected and the changes of some secondary bonds such as hydrogen bonds and salt bonds were observed. Detailed analysis of these spectra indicates that the alpha-helix of insulin molecule was destroyed after the exposure, which is proved by the shift of the peak of the amide I region toward higher wave number and by the appearance of several new peaks: 1561 and 1594 cm(-1). The disulfides were affected by the weaken alpha-helix, and their vibrational modes were changed. Meanwhile the hydrogen bonding between the dimer are broken down which leads to the increase in the peak intensities at 1002 and at 1602 cm(-1).

[Study on the EMF-temperature co-effects on protein conformation by fluorometry].

The bioelectromagnetic effects are wildly concerned for a long time, and related researches are conducted in all kinds of directions in recent years. The EMF-temperature co-effects are more interesting nowadays. By studying the effects of EMF co-operated with temperature on a protein, an irreversible protein denaturation is found under the processing of EMF, and this denaturation is also Arrhenius-rule-obeyed. In addition, a protein denaturation model under the EMF-temperature co-effects is built. In this paper the EMF-temperature co-effects are explained in a way of molecular reacting kinetics, and the athermal effects of EMF are also discussed to some degree.