CO2 laser therapy accelerates the healing of ulcers in the oral mucosa by inducing the expressions of heat shock protein-70 and tenascin C.

The treatment of ulceration or stomatitis with laser therapy is known to accelerate healing and relieve pain, but the underlying biological mechanism is not fully understood. The present study used a mouse model of ulceration to investigate the molecular mechanisms by which CO2 laser therapy accelerated the wound healing process. An ulcer was experimentally created in the palatal mucosa of the mouse and irradiated with light from a CO2 laser. Compared with controls (no irradiation), laser irradiation induced the proliferation of epithelial cells and faster re-epithelialization of the wound area. Immunohistochemistry experiments showed that heat shock protein-70 (HSP70) was expressed mainly in the epithelium of normal palatal tissue, whereas there was little tenascin C (TnC) expression in the epithelium and mesenchyme under normal conditions. Laser irradiation induced HSP70 mRNA and protein expression in the lamina propria as well as TnC expression in the mesenchyme underlying the renewing epithelium. Epithelial cells and fibroblasts were exposed to heated culture medium or laser irradiation to establish whether hyperthermia mimicked the effect of laser irradiation. Culture of fibroblasts in heated medium increased the expressions of both TnC and TGF-ß1, whereas laser irradiation induced only TnC expression. The present study indicates that CO2 laser irradiation exerts a photobiogenic effect to up-regulate TnC expression without inducing TGF-ß1 expression. We suggest that CO2 laser therapy has an advantage over thermal stimulation.

Effects of prolonged exposure to moderate static magnetic field and its synergistic effects with alkaline pH on Enterococcus faecalis.

Static magnetic field (SMF) has been shown to biologically affect various microorganisms, but its effects on Enterococcus faecalis, which is associated with multiple dental infections, have not been reported yet. Besides, Enterococcus faecalis was found to be resistant to the alkaline environment provided by a major dental antimicrobial, calcium hydroxide. Therefore, the antibacterial activity of prolonged exposure to moderate SMF (170 mT) and its possible synergistic activity with alkaline pH (pH = 9) were evaluated in the study. The ability to form a biofilm under these conditions was examined by crystal violet assay. Real-time quantitative PCR was performed to evaluate the relative expression of stress (dnaK and groEL) and virulence (efaA, ace, gelE and fsrC) related genes. As the results indicated, cell proliferation was inhibited after 120 h of SMF exposure. What's more, the combined treatment of SMF and alkaline pH showed significantly improved antimicrobial action when compared to single SMF and alkaline pH treatment for more than 24 h and 72 h respectively. However, the ability to form a biofilm was also enhanced under SMF and alkaline pH treatments. SMF can induce stress response by up-regulating the expression of dnaK and elevate virulence gene expression (efaA and ace). These responses were more significant and more genes were up-regulated including groEL, gelE and fsrC when exposed to SMF and alkaline pH simultaneously. Hence, combination of SMF and alkaline pH could be a promising disinfection strategy in dental area and other areas associated with Enterococcus faecalis infections.

[The cardiac injury effect of microwave radiation on rabbit and its mechanism].

OBJECTIVE: To investigate the cardiac injury effect of different intensities microwave radiation on rabbits and its possible mechanism. METHODS: Rabbits were radiated by intensity of 50, 100, 150 and 200 mW/cm2 2450 MHz microwave for 20 min. 6 h after microwave radiation, the heart tissue was taken. ATP and mitochondria complex IV and V were measured in myocardial cells. The changes of myocardial tissue were observed by light microscopic. The expression of Caspase-3 and HSP 70 were detected by western blotting. RESULTS: The activity of ATP and mitochondria complex IV and V decreased significantly compared with normal control in cardiac tissue. 100, 150 and 200 mW/cm2 microwave radiation group vs. control group (P <0. 05). The HE staining result showed that myocardial cell appears edema, muscle fiber malalignment, cells appeared obvious injury. Results of western blotting showed that the expression of Caspase-3 and HSP 70 protein increased significantly in different dosage radiation group (P <0. 05). CONCLUSION: Microwave radiation has injury effect on rabbit heart. The possible mechanism may be related with inducing cell apoptosis by changing of stress level in myocardial cell.

EMF radiation at 2450 MHz triggers changes in the morphology and expression of heat shock proteins and glucocorticoid receptors in rat thymus.

AIMS: Electromagnetic fields (EMFs) can act as inducers or mediators of stress response through the production of heat shock proteins (HSPs) that modulate immune response and thymus functions. In this study, we analyzed cellular stress levels in rat thymus after exposure of the rats to a 2.45 GHz radio frequency (RF) using an experimental diathermic model in a Gigahertz Transverse Electromagnetic (GTEM) chamber. MAIN METHODS: In this experiment, we used H&E staining, the ELISA test and immunohistochemistry to examine Hsp70 and Hsp90 expression in the thymus and glucocorticoid receptors (GR) of 64 female Sprague­Dawley rats exposed individually to 2.45 GHz (at 0, 1.5, 3.0 or 12.0 W power). The 1 g averaged peak and mean SAR values in the thymus and whole body of each rat to ensure that sub-thermal levels of radiation were being reached. KEY FINDINGS: The thymus tissue presented several morphological changes, including increased distribution of blood vessels along with the appearance of red blood cells and hemorrhagic reticuloepithelial cells. Levels of Hsp90 decreased in the thymus when animals were exposed to the highest power level (12 W), but only one group did not show recovery after 24 h. Hsp70 presented no significant modifications in any of the groups. The glucocorticoid receptors presented greater immunomarking on the thymic cortex in exposed animals. SIGNIFICANCE: Our results indicate that non-ionizing sub-thermal radiation causes changes in the endothelial permeability and vascularization of the thymus, and is a tissue-modulating agent for Hsp90 and GR.

Contribution of the immune system to bystander and non-targeted effects of ionizing radiation.

Considerable progress has recently been achieved in the understanding of molecular mechanisms involved in cellular radiation responses and radiation mediated microenvironmental communication. In line with that, it has become more and more obvious that X-irradiation causes distinct immunological effects ranging from anti-inflammatory activities if applied at low (<1 Gy) doses to harmful inflammatory side effects, radiation-induced immune modulation or induction of anti-tumour immune responses at higher doses. Moreover, experimental and clinical evidences indicate that these effects not only originate from direct nuclear damage but also include non-(DNA) targeted mechanisms including bystander, out of field distant bystander (abscopal) effects and genomic instability. The purpose of the present review is to elucidate immune responses that are initiated or affected by ionizing radiation, with a special emphasis on anti-inflammatory and abscopal effects and the induction of stress-induced anti-tumour immunity.

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields.

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti-sense orientation under the control of a heat-inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy. Previous findings suggest an alternative approach to inducing a heat shock response, specifically through the use of extremely low frequency (ELF) electrical field stimulation. To further pursue this approach, we investigated HSP70 responses in transfected rat primary fibroblast (RAT1) cells exposed to 10 Hz electric fields at intensities of 20-500 V/m. We confirmed that low frequency electric fields can induce HSP70 heat shock expression, with peak responses obtained at 8 h following a 2 h field exposure. However, the approximate threefold increase in expression is substantially lower than that obtained using thermal stimulation, raising questions of the clinical utility of the response.

Radiation combined with hyperthermia induces HSP70-dependent maturation of dendritic cells and release of pro-inflammatory cytokines by dendritic cells and macrophages.

PURPOSE: Hyperthermia (HT) treatment of cancer patients was revived over the last years and has been proven to be beneficiary for many cancer entities when applied temperature controlled in multimodal treatments. We examined whether a combination of ionizing irradiation (X-ray) and HT (41.5°C; 1 h) can induce the release of heat shock protein (HSP) 70 by tumor cells and thereby lead to the activation of dendritic cells and macrophages. MATERIAL AND METHODS: Extracellular HSP70 was detected in supernatants (SN) of treated colorectal tumor cells by ELISA. Maturation of dendritic cells (DC) after contact with the SN was measured by flow-cytometry. Phagocytosis assays were conducted to get hints about the immune stimulating potential of the tumor cells after the respective treatments. RESULTS: An increased surface expression of HSP70 was observed after X-ray or X-ray plus HT while the amount of extracellular HSP70 was only increased when HT was given additionally. A high up-regulation of the co-stimulation molecule CD80 and the chemokine receptor CCR7 on DC was measured after contact with SN of X-ray plus HT treated cells. This was dependent on extracellular HSP70. Combined treatments further led to significantly increased phagocytosis rates of macrophages and DC and increased pro-inflammatory cytokine (IL-8 and IL-12) secretion. CONCLUSION: X-ray combined with HT induces HSP70 dependent activation of immune cells and might generate a tumor microenvironment beneficial for cure.

Exposure to ELF-pulse modulated X band microwaves increases in vitro human astrocytoma cell proliferation.

Common concern about the biological effects of electromagnetic fields (EMF) is increasing with the expansion of X-band microwaves (MW). The purpose of our work was to determine whether exposure to MW pulses in this range can induce toxic effects on human astrocytoma cells. Cultured astrocytoma cells (Clonetics line 1321N1) were submitted to 9.6 GHz carrier, 90% amplitude modulated by extremely low frequency (ELF)-EMF pulses inside a Gigahertz Transversal Electromagnetic Mode cell (GTEM-cell). Astrocytoma cultures were maintained inside a GTEM-incubator in standard culture conditions at 37+/-0.1 degrees C, 5% CO2, in a humidified atmosphere. Two experimental conditions were applied with field parameters respectively of: PW 100-120 ns; PRF 100-800 Hz; PRI 10-1.25 ms; power 0.34-0.60 mW; electric field strength 1.25-1.64 V/m; magnetic field peak amplitude 41.4-54.6 microOe. SAR was calculated to be 4.0 x 10-4 W/Kg. Astrocytoma samples were grown in a standard incubator. Reaching 70-80% confluence, cells were transferred to a GTEM-incubator. Experimental procedure included exposed human astrocytoma cells to MW for 15, 30, 60 min and 24 h and unexposed sham-control samples. Double blind method was applied. Our results showed that cytoskeleton proteins, cell morphology and viability were not modified. Statistically significant results showed increased cell proliferation rate under 24h MW exposure. Hsp-70 and Bcl-2 antiapoptotic proteins were observed in control and treated samples, while an increased expression of connexin 43 proteins was found in exposed samples. The implication of these results on increased proliferation is the subject of our current research.

Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo.

The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

Dynamics and mechanism of HSP70 translocation induced by photodynamic therapy treatment.

Heat shock protein 70 (HSP70) is involved in nearly all intracellular compartments. It has been recently shown to be expressed on the outer cellular membrane under photodynamic therapy (PDT) treatment. However, the mechanism and function of HSP70 translocation to the cell surface during PDT treatment are not well understood. In this study, the dynamics and mechanism of HSP70 translocation onto the cell surface and its relationship with several key intracellular events after PDT treatment were investigated using confocal microscopy. HeLa and ASTC-a-1 tumor cells were treated by PDT using different doses. In the case of PDT-induced apoptosis, cytoplasmic HSP70 rapidly translocated to the cell surface after treatment, but it was not released into the medium. Such translocation was found to be dependent on the PDT dose. Moreover, during apoptosis, the translocation of HSP70 was closely related to the changes of mitochondrial transmembrane potential (DeltaPsim). Under non-lethal PDT induced surface stress, HSP70 also translocated to the cell surface, but with a slower rate and a lower final surface concentration. These findings reaffirm the HSP70 translocation onto the cell surface under PDT treatment in living cells. Our results also indicate that the function of the surface expression of HSP70, either initiated by mitochondrial disruption or direct surface stress, is to stabilize the plasma membrane integrity, although such function failed to prevent apoptosis induced by lethal PDT treatment, as evidenced in our study.

Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis.

PURPOSE: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis). MATERIALS AND METHODS: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5 degrees C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion. RESULTS: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines. CONCLUSION: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.

Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer.

The physiology and biochemistry behind the extreme tolerance to desiccation shown by the so-called anhydrobiotic animals represents an exciting challenge to biology. The current knowledge suggests that both carbohydrates and proteins are often involved in protecting the dry cell from damage, or in the repair of induced damage. Tardigrades belong to the most desiccation-tolerant multicellular organisms, but very little research has been reported on the biochemistry behind desiccation tolerance in this group. We quantified the induction of the heat-shock protein Hsp70, a very wide-spread stress protein, in response to desiccation, ionising radiation, and heating, in the anhydrobiotic tardigrade Richtersius coronifer using an immuno-westernblot method. Elevated levels of Hsp70 were recorded after treatment of both heat and ionising radiation, and also in rehydrated tardigrades after a period of desiccation. In contrast, tardigrades in the desiccated (dry) state had reduced Hsp70 levels compared to the non-treated control group. Our results suggest that Hsp70 may be involved in the physiological and biochemical system underlying desiccation (and radiation) tolerance in tardigrades, and that its role may be connected to repair processes after desiccation rather than to biochemical stabilization in the dry state.

Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins.

Effects of 50 Hz sinusoidal magnetic fields on Hsp27, Hsp70, Hsp90 expression in porcine aortic endothelial cells (PAEC).

The aim of the present study was to investigate the influence of 50 Hz sinusoidal magnetic field on Hsp27, Hsp70, and Hsp90 expression in a model of primary culture of porcine aortic endothelial cells (PAEC). We took into consideration the Hsp profile in terms of mRNA expression, protein expression and protein localization inside the cells. The choice of the cell system was motivated by the involvement of the endothelial cells in the onset of many diseases; moreover, only few reports describe the effects of extremely low frequency magnetic fields (ELF-MFs) on such cells. ELF-MF exposure induced an increase in the mRNA levels of the three proteins, which was statistically significant for Hsp70. On the contrary, we did not observe any influence on Hsp27, Hsp70, and Hsp90 protein levels. Analysis in situ by immunofluorescence revealed that ELF-MF exposure affected the cellular distribution of Hsp27; in particular a partial relocalization in the nucleus was observed.

Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation.

In this study, we have investigated if current cancer therapeutic modalities including hyperthermia and ionizing radiation can increase the expression of NKG2D ligands in human cancer cell lines. The expressions of NKG2D ligands were induced by both heat shock and ionizing radiation in various cell lines including KM12, NCI-H23, HeLa and A375 cells with peaks at 2 h and 9 h after treatment, respectively, although inducibility of each NKG2D ligand was various depending on cell lines. During the induction of NKG2D ligands, heat shock protein 70 was induced by heat shock but not by ionizing radiation. These results were followed by increased susceptibilities to NK cell-mediated cytolysis after treatment with heat shock and ionizing radiation. These results suggest that heat shock and ionizing radiation induce NKG2D ligands and consequently might lead to increased NK cell-mediated cytotoxicity in various cancer cells.

Human skin cell stress response to GSM-900 mobile phone signals. In vitro study on isolated primary cells and reconstructed epidermis.

In recent years, possible health hazards due to radiofrequency radiation (RFR) emitted by mobile phones have been investigated. Because several publications have suggested that RFR is stressful, we explored the potential biological effects of Global System for Mobile phone communication at 900 MHz (GSM-900) exposure on cultures of isolated human skin cells and human reconstructed epidermis (hRE) using human keratinocytes. As cell stress markers, we studied Hsc70, Hsp27 and Hsp70 heat shock protein (HSP) expression and epidermis thickness, as well as cell proliferation and apoptosis. Cells were exposed to GSM-900 under optimal culture conditions, for 48 h, using a specific absorption rate (SAR) of 2 W x kg(-1). This SAR level represents the recommended limit for local exposure to a mobile phone. The various biological parameters were analysed immediately after exposure. Apoptosis was not induced in isolated cells and there was no alteration in hRE thickness or proliferation. No change in HSP expression was observed in isolated keratinocytes. By contrast, a slight but significant increase in Hsp70 expression was observed in hREs after 3 and 5 weeks of culture. Moreover, fibroblasts showed a significant decrease in Hsc70, depending on the culture conditions. These results suggest that adaptive cell behaviour in response to RFR exposure, depending on the cell type and culture conditions, is unlikely to have deleterious effects at the skin level.

Effects of low-power laser radiation on mice immunity.

BACKGROUND/PURPOSE: Because of large interest in biological effects of laser radiation used in laser therapy, the effect of extremely low-level red laser light intensity on the immune cell activity has been studied in the animal model with well-characterized macrophage and T cell populations as responder cells producing cytokines, protective proteins, active oxygen, and nitric compounds. To study of the possible side effects of laser immunotherapy we monitored the productions of cytokines, nitric oxide (NO), and heat shock protein 70 (Hsp70) in mice subjected to a periodic laser exposure for 1 month. METHODS: Helium-neon laser radiation with the power of 0.2 mW/cm2 and wavelength of 632.8 nm was applied on two different mouse skin surfaces, i.e. a thymus projection area or a hind limb. Healthy NMRI male mice were irradiated repeatedly with laser light for 1 min with 48-h intervals for 30 days. The animals were divided into three groups of 25 mice. The first and the second groups were exposed to laser light, on the thymus and hind limb area, respectively. The third, sham-irradiated group served as a control. Early and prolonged effects of laser radiation on the levels of NO (by Griess assay), Hsp70 (by Western blot assay), tumor necrosis factors (TNF-alpha and TNF-beta) (by cytotoxic assay using L929 cells as targets), and interleukin-2 (IL-2) (by ELISA assay) were determined. RESULTS: The dynamics of immune responses to low-power laser light intensity was shown to be dependent on two factors, i.e. the cumulative dose and the localization of the irradiated surface. Besides, various populations of cells demonstrated different sensitivity to laser radiation, with T cells being more responsive among examined populations of the cells. Low intensity laser light induced an immune cell activity when the exposure duration did not exceed 10 days, while a more prolonged period of treatment generated more severe changes in the immune system, up to immunosuppression. The treatment of the thymus zone resulted in more pronounced changes in the cytokine production as well as in NO and Hsp70 synthesis. CONCLUSION: Low-power laser irradiation showed more effective immunomodulatory effects when applied on the thymus projection area. The rise in IL-2 and Hsp70 production related to a short-term effect of laser application may be reversed after repeating laser treatment. We suggest that for the support of immune system stability, the prolonged laser therapy should be accompanied by supplementary methods.

Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP.

Gp96 (GRP94) is a dimeric glycoprotein and is the endoplasmic reticulum representative of the hsp90 family of molecular chaperones. In addition to the protein substrates it chaperones, gp96 binds weakly to both peptides and ATP, and has been shown to self-assemble into discrete oligomers upon heat shock at 50 degrees C, although physiological roles for these phenomena have not been well established. Our studies indicate that gp96 homooligomerizes irreversibly in vitro at temperatures as low as 42 degrees C and could involve pre-dissociation of dimers to monomers. Oligomerization is inhibited significantly by ATP; hydrolysis is not required, since ADP, ATP-gamma-S, and NECA inhibit self-assembly equally well. Peptide ligands do not competitively inhibit gp96 self-assembly and, in fact, bind to all oligomeric species, including the dimer. Together, these findings suggest that (1) heat-enhanced chaperone activity does not reside in oligomers per se, and (2) the regions of gp96 involved in peptide binding and oligomerization are distinct.

Assessment of cellular response to thermal laser injury through bioluminescence imaging of heat shock protein 70.

Assessment of laser-induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser-induced injury. A stable cell line (NIH-3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium-yttrium aluminum garnet laser (lambda = 2.1 microm, taup = 250 micros, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum bioluminescence over a range of 500-700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8-12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant-temperature water bath and laser experiments were carried out.

50 Hz magnetic fields activate mussel immunocyte p38 MAP kinase and induce HSP70 and 90.

Fifty hertz magnetic fields (MFs) induced the expression of heat shock proteins (HSPs) 70 and 90 in immunocytes of the mussel Mytilus galloprovincialis. Animals exposed at 300 microT for three different times (30; 2 x 30; 3 x 30 min), did not show differences in the HSP densitometric values in comparison with non-exposed mussels. At 400 microT, exposed animals showed a time-dependent increase in HSP expression as revealed by Western blot. After exposure to 600 microT, the HSP densitometric values were significantly higher than controls but not related to exposure duration. The induction of HSPs is concomitant with the activation of p38 MAP kinase signalling pathway. The present findings suggest the possibility to modulate the expression of HSPs by an appropriate time-intensity magnetic field exposure.