Search

Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

Little information is available about the effects of exposure to pulsed microwaves on neuronal Ca2+ signaling under non-thermal conditions. In this study, rat pheochromocytoma (PC12) cells were exposed to pulsed microwaves for 6 min at a specific absorption rate (SAR) of 4 W/kg to assess possible real-time effects. During microwave exposure, free calcium dynamics in the cytosol, mitochondria, and nucleus of cells were monitored by time-lapse microfluorimetry using a genetically encoded calcium indicator (ratiometric-pericam, ratiometric-pericam-mt, and ratiometric-pericam-nu). We established a waveguide-based real-time microwave exposure system under accurately controlled environmental and dosimetric conditions and found no significant changes in the cytosolic, mitochondrial, or nuclear calcium levels in PC12 cells. These findings suggest that no dynamic changes occurred in [Ca2+]c, [Ca2+]m, or [Ca2+]n of PC12 cells at the non-thermal level., (Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure., (© 2017 International Society for Neurochemistry.)

Photobiomodulation (PBM) using red or near-infrared (NIR) light has been used to stimulate the proliferation and differentiation of adipose-derived stem cells. The use of NIR wavelengths such as 810nm is reasonably well accepted to stimulate mitochondrial activity and ATP production via absorption of photons by cytochrome c oxidase. However, the mechanism of action of 980nm is less well understood. Here we study the effects of both wavelengths (810nm and 980nm) on adipose-derived stem cells in vitro. Both wavelengths showed a biphasic dose response, but 810nm had a peak dose response at 3J/cm 2 for stimulation of proliferation at 24h, while the peak dose for 980nm was 10-100 times lower at 0.03 or 0.3J/cm 2 . Moreover, 980nm (but not 810nm) increased cytosolic calcium while decreasing mitochondrial calcium. The effects of 980nm could be blocked by calcium channel blockers (capsazepine for TRPV1 and SKF96365 for TRPC channels), which had no effect on 810nm. To test the hypothesis that the chromophore for 980nm was intracellular water, which could possibly form a microscopic temperature gradient upon laser irradiation, we added cold medium (4°C) during the light exposure, or pre-incubated the cells at 42°C, both of which abrogated the effect of 980nm but not 810nm. We conclude that 980nm affects temperature-gated calcium ion channels, while 810nm largely affects mitochondrial cytochrome c oxidase., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Copper(II) pyridoxal Schiff base complexes [Cu(L(1)/L(2))(B)]ClO4 (1-4), where HL(1) is 4-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol (in 1 and 2), HL(2) is 2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)phenol (in 3, 4), B is 11-(9-acridinyl)dipyrido[3,2-a:2',3'-c]phenazine (acdppz in 1 and 3), dipyrido[3,2-a:2',3'-c]phenazine (in 2) and 1,10-phenanthroline (in 4), were synthesized, characterized and their photocytotoxicity in visible light, intracellular localization, cellular uptake and DNA photocleavage activity were studied. Complex 4 was characterized by X-ray crystallography. Complexes 1 and 3 having acdppz as photosensitizer showed significant photocytotoxicity in visible light in HeLa and MCF7 cells giving IC50 value of <0.6 μM, while being relatively non-toxic in dark. The complexes were non-toxic to non-tumorigenic HPL1D cells both in light and dark conditions. Complex 1 showed significant localization in the cytoplasm of HeLa cells within 4 h of treatment, as evidenced from confocal microscopy. DCFDA assay on 1 suggested generation of intracellular reactive oxygen species in HeLa cells upon photo-exposure. Importantly, Annexin-V-FITC/PI assay indicated photo-induced apoptotic cell death., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies  ∼70 eV, substantially lower than that of liquid water  ∼78 eV. Monte Carlo simulations for 10-50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of  ∼1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

TRPV1 is a Ca2+ permeable channel and gated by noxious heat, oxidative stress and capsaicin (CAP). Some reports have indicated that non-ionized electromagnetic radiation (EMR)-induces heat and oxidative stress effects. We aimed to investigate the effects of distance from sources on calcium signaling, cytosolic ROS production, cell viability, apoptosis, plus caspase-3 and -9 values induced by mobile phones and Wi-Fi in breast cancer cells MCF-7 human breast cancer cell lines were divided into A, B, C and D groups as control, 900, 1800 and 2450 MHz groups, respectively. Cells in Group A were used as control and were kept in cell culture conditions without EMR exposure. Groups B, C and D were exposed to the EMR frequencies at different distances (0 cm, 1 cm, 5 cm, 10 cm, 20 cm and 25 cm) for 1h before CAP stimulation. The cytosolic ROS production, Ca2+ concentrations, apoptosis, caspase-3 and caspase-9 values were higher in groups B, C and D than in A group at 0 cm, 1 cm and 5 cm distances although cell viability (MTT) values were increased by the distances. There was no statistically significant difference in the values between control, 20 and 25 cm. Wi-Fi and mobile phone EMR placed within 10 cm of the cells induced excessive oxidative responses and apoptosis via TRPV1-induced cytosolic Ca2+ accumulation in the cancer cells. Using cell phones and Wi-Fi sources which are farther away than 10 cm may provide useful protection against oxidative stress, apoptosis and overload of intracellular Ca2+. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

More than 20% of the total caloric intake of human population comes from rice. The expression of rice genes and hence, the concentration of enzymatic proteins might vary due to several biotic and abiotic stresses. It in turn, can influence the overall metabolism and survivability of rice plant. Thus, understanding the rice cellular metabolism, its plasticity and potential readjustments under different perturbations can help rice biotechnologists to design efficient rice cultivars. Here, using the flux balance analysis (FBA) method, with the help of in-silico reaction deletion strategy, we study the metabolic plasticity of genome-scale metabolic model of rice leaf. A set of 131 reactions, essential for the production of primary biomass precursors is identified; deletion of any of them can inhibit the overall biomass production. Usability Index (IU) for the rest of the reactions are estimated and based on this parameter, they are classified into three categories-maximally-favourable, quasi-favourable and unfavourable for the primary biomass production. The lower value of 1 - IU of a reaction suggests that the cell cannot easily bypass it for biomass production. While some of the alternative paths are energetically equally efficient, others demand for higher photon. The variations in (i) ATP/NADPH ratio, (ii) exchange of metabolites through chloroplastic transporters and (iii) total biomass production are also presented here. Mutual metabolic dependencies of different cellular compartments are also demonstrated.

Developing pharmacological strategies for controlling ionizing radiation (IR)-induced cell death is important for both mitigating radiation damage and alleviating the side effects of anti-cancer radiotherapy manifested in surrounding tissue morbidity. Exposure to IR often triggers the onset of p53-dependent apoptotic pathways. Here we build a stochastic model of p53 induced apoptosis comprised of coupled modules of nuclear p53 activation, mitochondrial cytochrome c release and cytosolic caspase activation that also takes into account cellular heterogeneity. Our simulations show that the strength of p53 transcriptional activity and its coupling (or timing with respect) to mitochondrial pore opening are major determinants of cell fate: for systems where apoptosis is elicited via a p53-transcription-independent mechanism, direct activation of Bax by p53 becomes critical to IR-induced-damage initiation. We further show that immediate administration of PUMA inhibitors following IR exposure effectively suppresses excessive cell death, provided that there is a strong caspase/Bid feedback loop; however, the efficacy of the treatment diminishes with increasing delay in treatment implementation. In contrast, the combined inhibition of Bid and Bax elicits an anti-apoptotic response that is effective over a range of time delays.

Cytosolic alkalization has been shown to function as a key player in multiple stimuli-induced stomatal closure, but its role and relationship with hydrogen peroxide (H2O2) in ultraviolet B (UV-B)-induced stomatal closure remains unknown. In this paper, by stomatal bioassay and laser-scanning confocal microscopy, we observed that 0.5 W m(-2) UV-B induced cytosolic alkalization and H2O2 production in guard cells while inducing stomatal closure in Arabidopsis (Arabidopsis thaliana). Butyrate (a weak acid) reduced the cytosolic pH/H2O2 production and prevented stomatal closure by UV-B. Methylamine (a weak base) induced H2O2 production and stomatal closure while enhancing the cytosolic alkalization in guard cells under light alone. The rise in cytosolic pH of wild-type guard cells on exposure to UV-B was evident at 15 min and substantial at 45 min while H2O2 production started to largely increase after 60 min. The failure of UV-B-induced H2O2 production in AtrbohD/F guard cells did not affect the changes of guard cell pH during the first 60 min of UV-B radiation, but largely suppressed cytosolic alkalization after 60 min of UV-B radiation. These results indicate that cytosolic alkalization mediates UV-B-induced stomatal closure via activating H2O2 production and that H2O2 production can feedback-enhance cytosolic alkalization in Arabidopsis guard cells., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Gamma irradiation of red blood cell (RBC) concentrates is routinely used to prevent transfusion-associated graft-versus-host disease. So far, the effects of ionizing radiation on RBC structure and function and especially the proteome are not fully understood., Experimental Design: RBC concentrates were irradiated with 30 Gy and stored for 1 or 15 days at 4 ± 2°C. Following cell lysis and hemoglobin depletion, 2D-DIGE was used to examine the changes of the cytosolic RBC proteome. Significantly altered spots were analyzed using bottom-up proteomic approaches and selected marker proteins validated by western blotting., Results: Gamma irradiation was found to enhance conventional RBC storage lesions. Following 15 days of postirradiation storage, the abundances of a total of 27 spots were significantly altered and 3 out of 13 identified proteins were selected and validated as potential marker proteins for the assessment of irradiation-induced cytosolic RBC lesions., Conclusions and Clinical Relevance: Gamma irradiation and subsequent ex vivo storage according to the Council of Europe guidelines were found to affect RBC protein structures. The validated marker proteins can serve as a basis for the development of a screening assay to monitor the quality of irradiated RBC concentrates during ex vivo storage., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ca(2+) plays a pivotal role in nitric oxide (NO)-promoted stomatal closure. However, the function of Ca(2+) in NO inhibition of blue light (BL)-induced stomatal opening remains largely unknown. Here, we analyzed the role of Ca(2+) in the crosstalk between BL and NO signaling in Vicia faba L. guard cells. Extracellular Ca(2+) modulated the BL-induced stomatal opening in a dose-dependent manner, and an application of 5 μM Ca(2+) in the pipette solution significantly inhibited BL-activated K(+) influx. Sodium nitroprusside (SNP), a NO donor, showed little effect on BL-induced K(+) influx and stomatal opening response in the absence of extracellular Ca(2+), but K(+) influx and stomatal opening were inhibited by SNP when Ca(2+) was added to the bath solution. Interestingly, although both SNP and BL could activate the plasma membrane Ca(2+) channels and induce the rise of cytosolic Ca(2+), the change in levels of Ca(2+) channel activity and cytosolic Ca(2+) concentration were different between SNP and BL treatments. SNP at 100 μM obviously activated the plasma membrane Ca(2+) channels and induced cytosolic Ca(2+) rise by 102.4%. In contrast, a BL pulse (100 μmol/m(2) per s for 30 s) slightly activated the Ca(2+) channels and resulted in a Ca(2+) rise of only 20.8%. Consistently, cytosolic Ca(2+) promoted K(+) influx at 0.5 μM or below, and significantly inhibited K(+) influx at 5 μM or above. Taken together, our findings indicate that Ca(2+) plays dual and distinctive roles in the crosstalk between BL and NO signaling in guard cells, mediating both the BL-induced K(+) influx as an activator at a lower concentration and the NO-blocked K(+) influx as an inhibitor at a higher concentration., (© 2013 Institute of Botany, Chinese Academy of Sciences.)

Reactive oxygen species play a key role in the response of plants to abiotic stress conditions. Their level is controlled in Arabidopsis thaliana by a large network of genes that includes the H(2)O(2)-scavenging enzymes cytosolic ascorbate peroxidase (APX) 1 and 2. Although the function of APX1 has been established under different growth conditions, genetic evidence for APX2 function, as well as for the mode of cooperation between APX1 and APX2, is very limited. This study characterized the response of Arabidopsis mutants deficient in APX1, APX2, and APX1/APX2 to heat, salinity, light, and oxidative stresses. The findings reveal that deficiency in APX2 resulted in a decreased tolerance to light stress, as well as an enhanced tolerance to salinity and oxidative stresses. Interestingly, plants lacking APX2 were more sensitive to heat stress at the seedling stage, but more tolerant to heat stress at the reproductive stage. Cooperation between APX1 and APX2 was evident during oxidative stress, but not during light, salinity, or heat stress. The findings demonstrate a role for APX2 in the response of plants to light, heat, salinity, and oxidative stresses. The finding that plants lacking APX2 produced more seeds under prolonged heat stress conditions suggests that redundant mechanisms activated in APX2-deficient plants during heat stress play a key role in the protection of reproductive tissues from heat-related damage. This finding is very important because heat-associated damage to reproductive tissues in different crops is a major cause for yield loss in agriculture production worldwide.

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

In many recent publications, supposed athermal effects of water-filtered infrared A (wIRA) irradiation are discussed. Those effects are mainly attributed to wavelengths in the range from 780 to 1440 nm, and should not result from warming of cellular water or any aqueous medium surrounding the irradiated sample caused by wIRA absorption. Athermal effects are considered to be induced directly by absorption of different wavelengths of the wIRA spectrum by cellular molecules or structures except water. To distinguish between thermal and athermal effects, irradiated samples have to be subjected to a very effective and precise temperature homeostasis. Any experimental effects can only be attributed to pure athermal effects, if the temperature of the irradiated samples is verifiably constant and does not result in hyperthermia. Here, data of temperature distribution in Petri dishes of different types filled with aqueous medium are presented which were estimated by model calculation for different setups of cooling. Additionally, the real temperature development was directly measured. Such a cooling unit enables long-term application of high wIRA irradiances and large doses without any detectable warming of the irradiated samples, in single cell layers. Using such a setup, thermal and athermal effects can be compared and in addition to that quantified., (© 2011 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Glutathione (GSH) is involved in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we examined the effects of GSH-decreasing chemicals, p-nitrobenzyl chloride (PNBC), iodomethane (IDM), and ethacrynic acid (EA), on ABA- and MeJA-induced stomatal closure in Arabidopsis. Treatments with PNBC, IDM, and EA decreased GSH contents in guard cells. Depletion of GSH by PNBC and IDM enhanced ABA- and MeJA-induced stomatal closure and inhibition of light-induced stomatal opening by ABA, whereas EA did not enhance either ABA- and MeJA-induced stomatal closure or inhibition of light-induced stomatal opening by ABA. Depletion of GSH did not significantly increase the production of the reactive oxygen species (ROS), cytosolic alkalization, or cytosolic Ca(2+) oscillation induced by ABA and MeJA. These results indicate that depletion of GSH enhances ABA- and MeJA-induced stomatal closure without affecting ROS production, cytosolic alkalization, or cytosolic Ca(2+) oscillation in guard cells of Arabidopsis.

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K(+)-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca(2+) and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2',7'-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.

Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B(6)) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response., (© Springer-Verlag 2010)

The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes, and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules, in many cases, internalization to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic release of the therapeutic molecule is the membrane-barrier of the endocytic vesicles. Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides, and the chemotherapeutic bleomycin. PCI has also been shown to enhance the treatment effect of targeted therapeutic macromolecules. The present protocol describes PCI of an epidermal growth factor receptor (EGFR)-targeted protein toxin (Cetuximab-saporin) linked via streptavidin-biotin for screening of targeted toxins as well as PCI of nonviral polyplex-based gene therapy. Although describing in detail PCI of targeted protein toxins and DNA polyplexes, the methodology presented in these protocols are also applicable for PCI of other gene therapy vectors (e.g., viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), polymers, nanoparticles, and some chemotherapeutic agents.

The aim of this study was to investigate the influence of 50 Hz magnetic or static magnetic fields of 0.5 mT on subsets of human CD4(+) T cells in terms of cytokine release/content, cell proliferation and intracellular free calcium concentration. CD4(+) T cells can be divided into different subsets on the basis of surface marker expression, such as CD45, and T cells can be divided into naive (CD45RA(+)) and memory (CD45RA(-)) cells. In this study, the effects of magnetic fields after 24 and 48 h of cell culture were analyzed. We found that the CD4(+)CD45RA(-) T subset were more sensitive after 2 h of exposure. Decreases in the release/content of IFN-gamma, in cell proliferation and in intracellular free calcium concentrations were observed in exposed CD4(+)CD45RA(-) T cells compared to CD4(+)CD45RA(+) T cells. The results suggest that exposure to the magnetic fields induces a delay in the response to stimulants and that modifications are rapidly reversible, at least after a short exposure.

Calcium ions are involved in a number of important signal transduction pathways in cells. Cytosolic calcium concentration ([Ca(2+)](c)) can be affected by the activation of Ca(2+) channels through the action of ligands such as ATP. The response of [Ca(2+)](c) to ligands may be affected by external factors like magnetic fields. The purpose of this study was to determine if exposure to a static magnetic field (SMF) for 800 s altered the [Ca(2+)](c) response to ATP in undifferentiated HL-60 cells. We sham exposed or field exposed fura-2 loaded HL-60 cells to a SMF of 1, 10, and 100 mT. Cells were activated with ATP 300 s into the exposure. The level of [Ca(2+)](c) was followed before, during, and after field or sham exposure with a ratiometric fluorescence spectroscopy system. It was found that high concentrations of ATP resulted in greater [Ca(2+)](c) responses, but faster recovery to near basal levels. The application of 1, 10, or 100 mT SMF did not affect the [Ca(2+)](c) response to ATP. Future work could examine the effect of a longer SMF exposure on the [Ca(2+)](c) response to ATP. Longer exposures might provide sufficient time for morphological changes in the plasma membrane to occur., (Copyright 2008 Wiley-Liss, Inc.)

Based on the model of the two calcium stores developed by Goldbeter, and the viewpoint that the cell membrane is a major site of interaction for extremely-low-frequency (ELF) electromagnetic fields, the permeability of ions can be changed by electromagnetic fields, a theoretical model of cytosolic calcium oscillations influenced by magnetic fields is presented. It is shown that, the field frequency condition will influence the pattern of calcium oscillation while the field strength was given, and the magnetic field strength will change the pattern of calcium oscillation when the frequency was 50Hz and 100Hz. Therefore, intracellular calcium oscillations can be influenced by ELF magnetic fields and then a series cellular response could be transformed.

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt). Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca(2+)](cyt), and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca(2+)](cyt) rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca(2+)](cyt). Oscillations of [Ca(2+)](cyt) and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca(2+)](cyt) and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca(2+)](cyt) and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.

[6]-Gingerol, a naturally occurring plant phenol, is one of the major components of fresh ginger (Zingiber officinale Roscoe, Zingiberaceae) and has diverse pharmacologic effects. Here, we describe its novel anti-oxidant, anti-apoptotic, and anti-inflammatory activities in vitro and in vivo. In vitro, pre-treatment with [6]-gingerol reduced UVB-induced intracellular reactive oxygen species levels, activation of caspase-3, -8, -9, and Fas expression. It also reduced UVB-induced expression and transactivation of COX-2. Translocation of NF-kappaB from cytosol to nucleus in HaCaT cells was inhibited by [6]-gingerol via suppression of IkappaBalpha phosphorylation (ser-32). Examination by EMSAs and immunohistochemistry showed that topical application of [6]-gingerol (30 microM) prior to UVB irradiation (5 kJ/m(2)) of hairless mice, also inhibited the induction of COX-2 mRNA and protein, as well as NF-kappaB translocation. These results suggest that [6]-gingerol could be an effective therapeutic agent providing protection against UVB-induced skin disorders.

Extremely low frequency magnetic fields (ELF MF) have been reported to alter a number of cell signaling pathways, including those involved in proliferation, differentiation and apoptosis where cytosolic free calcium ([Ca(2+)](c)) plays an important role. To better understand the biological conditions under which ELF MF exposure might alter [Ca(2+)](c), we measured [Ca(2+)](c) by ratiometric fluorescence spectrophotometry during exposure to ELF MF in Jurkat E6.1 cells synchronized to different phases of the cell cycle. Suspensions of cells were exposed either to a near zero MF (Null) or a 60 Hz, 100 microT sinusoidal MF superimposed upon a collinear 78.1 microT static MF (AC + DC). An initial series of experiments indicated that the maximum increase in [Ca(2+)](c) above baseline after stimulation with anti-CD3 was significantly higher in samples exposed to AC + DC (n = 30) compared to Null (n = 30) with the largest difference in G2-M enriched samples. However, in a second study with G2-M enriched cells, samples treated with AC + DC (n = 17) were not statistically different from Null-treated samples (n = 27). Detailed analysis revealed that the dynamics in [Ca(2+)](c) before and after stimulation with anti-CD3 were dissimilar between Null samples from each study. From the results, we concluded (i) that the ELF MF increased [Ca(2+)](c) during an antibody-induced signaling event, (ii) that the ELF MF effect did not depend to a large degree on cell cycle, and (iii) that a field-related change in [Ca(2+)](c) signaling appeared to correlate with features in the [Ca(2+)](c) dynamics. Future work could evaluate [Ca(2+)](c) dynamics in relation to the phase of the cell cycle and inter-study variation, which may reveal factors important for the observation of real-time effects of ELF MF on [Ca(2+)](c).

Epidemiological and experimental evidences have established solar ultraviolet (UV) radiation as the leading cause of skin cancers. Specifically, the frequency of non-melanoma skin cancer, one of the malignancies with the most rapidly increasing incidence, is directly related to the total exposure to solar UV light. As part of a general effort to elucidate the components of cellular signal transduction pathways, the mechanisms of cellular responses to UV radiation have received considerable attention over the last few years. These efforts were driven mainly by the conviction that understanding how normal cells respond to extracellular stimuli such as exposure to UV radiation will undoubtedly help in deciphering what goes wrong in a variety of clinical disorders including skin cancers and will assist in the development of novel therapeutic strategies. Studies over the last decade have established that UV radiation induces a bewildering array of signal transduction pathways, some of which could lead to apoptotic cell death. UV-induced cell death by apoptosis is considered to be a natural protective mechanism that removes damaged keratinocytes and circumvents the risk of malignant transformation. In this review, we summarize some of the most important findings regarding the response and role of mitogen-activated protein kinases in UVA and UVB radiation-induced signaling to apoptosis in keratinocytes. We will also briefly discuss what is known about the role of the BCL-2 family of proteins, the emerging role of lysosomal proteases and other important cytosolic signaling proteins in UV-induced apoptosis.

The concentration of free cytosolic Ca2+ ([Ca2+]i), 45Ca2+ entry and the level of reduced glutathione (GSH) after x-irradiation in a dose of 4.5 Gy or 0.1 mM H2O2-treatment were investigated in isolated rat thymocytes during the period preceding electrophoretically detected DNA intranucleosomal fragmentation. Using fura-2 it was shown that the level of [Ca2+]i in X-irradiated thymocytes was not changed as compared with the control, while the GSH content was increased. The gradual increase in [Ca2+]i along with GSH level falling was detected in the H2O2-treated cells. 45Ca2+ entry in the cells exposed to apoptogenic stimuli was not enhanced. After addition of H2O2 to the cells previously treated with thapsigargin further [Ca2+]i increase in both normal and nominally calcium-free medium was detected. Cyclosporine A inhibited Ca2+-mobilizing effect of H2O2, but did not prevent it completely. The role of intracellular calcium depots in calcium homeostasis disturbance during oxidative stress and apoptosis is discussed.

The phytochrome (phy) family of sensory photoreceptors (phyA to phyE in Arabidopsis) enables plants to optimize their growth and development under natural light environments. Subcellular localization studies have shown that the photoreceptor molecule is induced to translocate from cytosol to nucleus by light, but direct evidence of the functional relevance of this translocation has been lacking. Here, using a glucocorticoid receptor-based fusion protein system, we demonstrate that both photoactivation and nuclear translocation combined are necessary and sufficient for the biological function of phyB. Conversely, neither artificial nuclear translocation of non-photoactivated phyB nor artificial retention of photoactivated phyB in the cytosol provides detectable biological activity. Together these data indicate that signal transfer from photoactivated phyB to its primary signaling partner(s) is localized in the nucleus, and conversely suggest the absence of a cytosolic pathway from photoactivated phyB to light-responsive genes.

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m(2), but not at 150 J/m(2), induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m(2) UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m(2) UVB, but not at 150 J/m(2). However, with both 150 and 500 J/m(2) UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m(2) UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.

The light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatases (FBPase) was investigated using enzyme activity assay, immunoblot, and Northern blot analyses. The enzyme activities of both chloroplast and cytosolic FBPases gradually increased under continuous white light illumination, although the increase in chloroplast FBPase was more drastic. Northern and immunoblot analyses also indicated that light stimulated the expression of both enzymes. Enzyme activity and the transcript levels of both enzymes gradually decreased under the dark treatment, although protein levels were unchanged for up to 24 h following the initiation of culture in the dark, indicating that reversible modifications of the enzymes may occur during the transition from light to dark (or the reverse). Light pulse experiments using blue (420 nm) and red/far-red (660/730 nm) light were carried out to analyze the photoreceptors related to the light-mediated expression of both enzymes. Expression of the chloroplast enzyme was very sensitive to red or far-red light pulses-it was induced by red light, but suppressed by far-red light pulses, as determined by enzyme activity, immunoblot, and Northern blot analyses, suggesting that red light signaling is involved in the control of chloroplast FBPase expression. However, cytosolic FBPase was virtually insensitive to blue or red/far-red light pulses in terms of enzyme activity, as determined by protein and transcript levels, indicating that cytosolic enzyme expression is not directly regulated by light signals. Instead, the expression of the cytosolic enzyme may be closely related to photosynthetic energy conversion accompanied by continuous white light irradiation.

Nuclear factor (NF)-kappa B pathways are influenced by the intracellular reduction-oxidation (redox) balance. While NF-kappa B is activated through inhibitor (I)-kappa B degradation by oxidative stress, its DNA binding is accelerated in the reduced state. We found that N,N'-diacetyl-L-cystine dimethylester (DACDM) suppressed the UVB-induced NF-kappa B binding activity at a much lower concentration (50-100 microM) than N-acetyl-L-cysteine (NAC, 10-30 mM). While NAC suppressed the I-kappa B degradation but not the DNA binding, DACDM prevented the activated NF-kappa B from binding DNA, without influencing the I-kappa B degradation. These properties of DACDM make it possible to effectively regulate the intracellular redox balance.

Reported changes in the cytosolic calcium concentration ([Ca2+](c)) as a result of exposure to extremely low frequency (ELF) magnetic fields (MF) have been equivocal. In this study, we examine the possibility that some of these differences are attributable to variability associated with the cell cycle, pH of the suspension medium, and response to a calcium agonist. We used a custom designed spectrofluorimeter to measure [Ca2+](c) in Indo 1-AM loaded Jurkat E6.1 cells suspended in conditioned RPMI 1640 medium containing 10% fetal bovine serum. Four exposures were examined: zero static MF (Null), 60 Hz 100 microT(peak) sinusoidal MF (AC), 78 microT static MF (DC), and the combination of the 60 Hz and the 78 microT static MF (AD + DC). A significant decrease in normalized [Ca2+](c) values between 375-495 s for the DC and AC + DC groups was found in comparison to the Null group. However, statistical analysis indicated that cell cycle and quality of the alpha-CD3 monoclonal antibody response were significant covariates, while pH was not a significant covariate. When the effect of these covariates was taken into account, all exposure groups were significantly different from the control. Our results suggest that ELF MF effects may not be seen unless correction is made for biological variability of each cell preparation with respect to cell cycle and [Ca2+](c) response to antigen stimulation., (Copyright 2002 Wiley-Liss, Inc.)

The influence of acute and chronic gamma irradiation (0.25 and 0.5 Gy doses) on rat lymphocytes was examined on the 1st and 10th days after irradiation. Acute gamma irradiation increased the intracellular calcium concentration in the cytoplasm of the lymphocytes on the 1st day after the influence of the radiation. Calcium concentration returned to the control level on the 10th day after the effect of the radiation at all the doses. In the lymphocytes after acute irradiation the increase of the Ca2+ concentration in the intracellular depots on the 1st day was observed. Changes were also observed in the physicochemical state of the plasma membrane of these cells. Research on the influence of prolonged gamma irradiation on intracellular calcium homeostasis revealed opposite effects in comparison with acute gamma irradiation.

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.

Natural phenolic compounds were tested in vitro for their effect on the activity of protein kinase C (PKC) isolated from the liver cytosol and the particulate fraction of unirradiated mice and mice irradiated at 5 Gy. Following irradiation, the PKC activity was found to be increased in both cytosolic and particulate fractions. Curcumin, ellagic acid and quercetin were effective in inhibiting radiation-induced PKC activity. Curcumin and ellagic acid were found to be more inhibitory towards radiation-induced PKC activity, while quercetin was the least effective. Curcumin was found to inhibit the activated cytosolic and particulate PKC at very low concentrations. Activation of PKC is one of the means of conferring radioresistance on a tumour cell. Suppression of PKC activity by phenolics may be one of the means of preventing the development of radioresistance following radiotherapy.

Whole body exposure of male rats to 7 Gy gamma irradiation increased lipid peroxidation in the liver resulting in biomembrane damage of subcellular structures and release of their enzymes. This is evidenced by increase of thiobarbituric acid-reactive substances (TBARS) in mitochondria, lysosomes and microsomes. This was associated with a decrease in activity of the enzymes specific for each subcellular fraction; namely, mitochondrial glutamate dehydrogenase (GDH), lysosomal beta-glucuronidase and microsomal glucose 6-phosphatase. This was paralleled by an increased activity of these enzymes in the cytosol. Rats were supplemented with lycopene, a carotenoid present in tomatoes (5 mg/kg weight/day), by gavage, for 7 days before exposure to 7 Gy gamma irradiation. This resulted in diminishing amount of TBARS recorded for each subcellular structure in the liver of irradiated animals. Significant amelioration in the decrease recorded for the activity of mitochondrial glutamate dehydrogenase, lysosomal beta-glucuronidase and microsomal glucose 6-phosphatase was observed. This was associated with significant amelioration in the increase recorded for the activity of these enzymes in the cytosol. It is postulated that lycopene could play an important role in the recovery of the integrity of biological membranes of the liver after radiation injury.

The ability to modulate the sensitivity of mammalian cells to ionizing radiation (IR) (e.g. using chemotherapeutics) is dependent on our understanding of the primary target and biochemical pathway that leads to IR-induced apoptosis. We demonstrate using a cell free assay that irradiation of mitochondria is a primary event that initiates IR-induced apoptosis. IR results in loss of mitochondrial membrane potential, opening of the permeability transition pore (PTP) and the release of cytochrome c (cyto c). Apaf-1 and ATP were required to initiate apoptosis upon release of cyto c from mitochondria. The importance of mitochondrial events in the initiation of IR-induced apoptosis was also supported by the observation that inhibition of caspase-9 by the over-expression of dominant negative mutants resulted in the inhibition of IR-induced apoptosis. In contrast, inhibition of caspase-8 had only a minor impact on IR-induced apoptosis. Over-expression of Bcl-X(L) inhibited the initiation of IR-induced apoptosis due to its ability to prevent the loss of mitochondrial membrane potential, PTP opening and cytochrome c release. In a cell free assay for apoptosis, mitochondria as well as cytosol derived from Bcl-X(L) over-expressing cells were less efficient at supporting apoptosis in response to IR suggesting multiple roles for Bcl-X(L) in the regulation of apoptosis.

Ionizing radiation in doses of 0.5 and 1.0 Gy modifies cytosol estrogen- and progestin-receptor complexes decreasing their acception by hepatocyte nuclei in the liver of gamma-irradiated female rats.

Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization. These events are not induced by mitochondria-independent apoptotic stimuli, such as Fas. Activation of cytosolic caspases by cytC in vitro is minimal at neutral pH, but maximal at acidic pH, indicating that mitochondria-induced acidification of the cytosol may be important for caspase activation; this finding is supported by results obtained from cells using protonophores. Cytosol acidification and cytC release are suppressed by oligomycin, a FoF1-ATPase/H +-pump inhibitor, but not by caspase inhibitors. Ectopic expression of Bax in wild-type, but not FoF1/H+-pump-deficient, yeast cells similarly results in mitochondrial matrix alkalinization, cytosol acidification and cell death. These findings indicate that mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis.

As a result of total chronic gamma irradiation of mice (137Cs, 0.6 cGy/day, 9 days) the functioning of superoxide generation and utilisation systems in liver were disturbed. The regulatory links between the activities of superoxide dismutase and glutathione peroxidase are found to be maintained. Postradiation effects were more expressed for a total dose of 1.2 cGy than for a dose of 5.4 cGy, providing support for the hypothesis of delayed reparation as a reason of harmful action of low-dose irradiation.

The present study was designed to test the hypothesis that a 60 Hz electromagnetic field could affect the influx of calcium ions across the plasma membrane through the so-called capacitative calcium entry system. Recordings of cytosolic calcium-ion concentrations in SV40-transformed Swiss 3T3 cells were obtained in real time during exposure to magnetic fields ranging from 0.3-50 mT or to sham conditions using the calcium-sensitive photoprotein aequorin. This was done for cell populations whose capacitative entry system was activated by either bradykinin or thapsigargin under a variety of experimental conditions. No effects of the magnetic field were observed on bradykinin-induced calcium transients and, with the exception of a small but statistically significant increase observed in experiments performed at 50 mT, no effects of the fields were observed on baseline calcium levels prior to or after such transients. The magnetic fields also had no effects on the size or kinetics of any of the thapsigargin-induced calcium transients. Overall, the data fail to support the hypothesis tested in this work.

The ability of Ukrain, a cytostatic and immunomodulating semisynthetic compound of thiophosphate-modified alkaloids of Chelidonium majus L., to modify the effects of irradiation on intracellular glucocorticoid reception in female rat liver was evaluated after intraperitoneal administration of the drug at 0.4 mg/kg of body weight. Ukrain caused the normalization of the intracellular glucocorticoid-receptor system, influenced by short-term whole-body gamma-irradiation of rats with 1 Gy, beginning on the 10th day after administration of the drug. It was found that Ukrain minimized the consequences of irradiation in the endocrine system of the experimental animals.

Enzyme, Western blot, and immunohistochemical analyses indicated that rat skin cytosol contained no detectable level of the homodimeric, alpha-class glutathione S-transferase (rGST) A4-4 which catalyzes the GSH conjugation of the toxic product, 4-hydroxy-2(E)-nonenal (HNE), nonenzymatically formed from n-6 polyunsaturated fatty acid residues of lipids by lipid peroxidation. Rats irradiated by single doses (4000-24,000 mJ/cm(2)) of ultraviolet B-band light (UVB, 200 mJ/cm(2)/min) markedly expressed rGSTA4-4 in the skin at a level one-fifth that of the liver in apparent specific activity toward HNE at a single dose of 24,000 mJ/cm(2). Skin rGSTA4-4 was isolated, purified to homogeneity, and identified with hepatic rGSTA4-4 by reverse-phase partition HPLC and by amino acid sequence analysis of its CNBr fission peptides. Immunohistochemistry with polyclonal antibody raised against rGSTA4-4 demonstrated the selective expression of rGSTA4-4 in epidermis and sebaceous glands localized in dermis after UVB irradiation., (Copyright 1999 Academic Press.)

1. The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo-1, or indo-1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2. Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via alpha-adrenergic receptors and the guanosine trisphosphate (GTP) binding protein-coupled phosphoinositide pathway. 3. Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA-induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 +/- 0.3 % (n = 6). 4. When Ca2+ was directly released from the inositol 1,4, 5 trisphosphate (IP3)-sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 +/- 0.5 % (n = 6). Thus, NA-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release. 5. Large and rapid elevation of [Ca2+]i (>15 microM) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in approximately 100 ms and a slow burst that continued for many seconds. 6. The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 +/- 0.1 % (n = 14). 7. We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.