Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization.

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.

Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis.

Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.

Inhibition of store-operated calcium entry in human lymphocytes by radiation: protection by glutathione.

The influence of gamma radiation on basal compared to activation-dependent Ca(2+) influx in human lymphocytes was investigated. A new quantitative fluorescence technique termed differential ratiometric fluorescence spectroscopy (DRFS) was employed. DRFS facilitated the real-time detection of changes in fluorescence in experimental and control cell samples simultaneously, enabling the resolution of acute moderate changes ( congruent with10-30%) in Ca(2+) (manganese) influx after exposure to ionizing radiation and other oxidant interventions. Exposure to radiation inhibited thapsigargin-stimulated store-operated Ca(2+) influx but not basal Ca(2+) influx in Jurkat T cells and human peripheral blood lymphocytes. The response of store-operated Ca(2+) influx to gamma radiation was dependent on dose between 5 and 40 Gy and was inhibited by preincubation with the Ca(2+) channel blocker Ni(2+), as determined with Jurkat T cells. Elevation of the intracellular concentration of glutathione significantly reduced the inhibition of Ca(2+) influx by gamma radiation. Similar to radiation, both the superoxide anion-generating xanthine/xanthine oxidase system and hydrogen peroxide inhibited thapsigargin-stimulated Ca(2+) influx in Jurkat T cells, and this inhibition was reversed in the presence of the antioxidant N-acetyl-l-cysteine. In conclusion, (1) ionizing radiation inhibited store-operated Ca(2+) entry in human lymphocytes, (2) the sensitivity of Ca(2+) influx to radiation was strictly dependent on depletion of Ca(2+) stores, and (3) glutathione protected against the inhibition of store-operated Ca(2+) entry by gamma radiation.

Increase in radiation-induced HPRT gene mutation frequency after nonthermal exposure to nonionizing 60 Hz electromagnetic fields.

It is widely accepted that moderate levels of nonionizing electric or magnetic fields, for example 50/60 Hz magnetic fields of about 1 mT, are not mutagenic. However, it is not known whether such fields can enhance the action of known mutagens. To explore this question, a stringent experimental protocol, which included blinding and systematic negative controls, was implemented, minimizing the possibility of observer bias or experimental artifacts. As a model system, we chose to measure mutation frequencies induced by 2 Gy gamma rays in the redox-sensitive hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster ovary cells. We tested whether a 12-h exposure to a 60 Hz sinusoidally oscillating magnetic-flux density (Brms = 0.7 mT) could affect the mutagenic effects of ionizing radiation on the HPRT gene locus. We determined that the magnetic-field exposure induced an approximate 1.8-fold increase in HPRT mutation frequency. Additional experiments at Brms = 0.23 and 0.47 mT revealed that the effect was reduced at lower flux densities. The field exposure did not enhance radiation-induced cytotoxicity or mutation frequencies in cells not exposed to ionizing radiation. These results suggest that moderate-strength, oscillating magnetic fields may act as an enhancer of mutagenesis in mammalian cells.

Magnetic field perturbations as a tool for controlling enzyme-regulated and oscillatory biochemical reactions.

The feasibility of magnetic field perturbations as a tool for controlling enzyme-regulated and oscillatory biochemical reactions is studied. Our approach is based on recent experimental results that revealed magnetic field effects on the in vitro activity of enzyme systems in accordance with the radical pair mechanism. A minimum model consisting of two coupled enzyme-regulated reactions is discussed that combines, in a self-consistent manner, magnetic field-sensitive enzyme kinetics with non-linear dynamical principles. Furthermore, a simple detector mechanism is described that is capable of responding to an oscillatory input. Results reveal that moderate-strength magnetic fields (B=1-100 mT) may effectively alter the dynamics of the system. In particular, a response behavior is observed that depends on: (1) the combination of static and time-varying magnetic fields; (2) the field amplitude; and (3) the field frequency in a non-linear fashion. The specific response behavior is critically determined by the biochemical boundary conditions as defined by the kinetic properties of the system. We propose an experimental implementation of the results based on the oscillatory peroxidase-oxidase reaction controlled by the enzyme horseradish peroxidase.

Model for magnetic field effects on radical pair recombination in enzyme kinetics.

A prototypical model for describing magnetic field effects on the reaction kinetics of enzymes that exhibit radical pair recombination steps in their reaction cycle is presented. The model is an extended Michaelis-Menten reaction scheme including an intermediate enzyme-substrate complex where a spin-correlated radical pair state exists. The simple structure of the scheme makes it possible to calculate the enzyme reaction rate explicitly by combining chemical kinetics with magnetic field-dependent spin kinetics (radical pair mechanism). Recombination probability is determined by using the exponential model. Simulations show that the size of the magnetic field effect depends on relations between different rate constants, such as 1) the ratio between radical pair-lifetime and the magnetic field-sensitive intersystem crossing induced by the hyperfine interaction and the delta g mechanisms and 2) the chemical rate constants of the enzyme reaction cycle. An amplification factor that is derived from the specific relations between the rate constants is defined. It accounts for the fact that although the magnetic field-induced change in radical pair recombination probability is very small, the effect on the enzyme reaction rate is considerably larger, for example, by a factor of 1 to 100. Model simulations enable a qualitative comparison with recent experimental studies reporting magnetic field effects on coenzyme B12-dependent ethanolamine ammonia lyase in vitro activity that revealed a reduction in Vmax/KM at low flux densities and a return to the zero-field rate or an increase at high flux densities.

Activation-dependent and biphasic electromagnetic field effects: model based on cooperative enzyme kinetics in cellular signaling.

Experiments on filed exposure effects of extremely-low-frequency electric and magnetic fields (EMFs) on biological systems have shown that, in many cases, the biological-functional status is of fundamental importance for an effective interaction. For example, studies of calcium uptake regulation in cells of the immune system, particularly in T lymphocytes, have revealed that, depending on the degree of cellular activation, either stimulatory, inhibitory, or no field exposure effects are observed for identical field parameters. A brief summary of the experimental findings is given, and a theoretical approach is presented that accounts in a qualitative manner for EMF exposure effects 1) that depend on the degree of cellular activation and 2) that exhibit a biphasic response behavior (stimulation/ inhibition). In the model, biochemical stimulation of the cell results in activation of specific signaling pathways that regulate calcium dynamics in the cell (calcium release from intracellular calcium stores and capacitative calcium entry). We assume that, controlled by these pathways, a specific EMF-sensitive enzyme system becomes activated. The activated enzyme, in turn, exhibits a feedback control on the signal processes, thus leading to a modulation of calcium entry. This modulation may affect other cellular processes that are calcium dependent (e.g., DNA synthesis). Magnetic field exposure is assumed to alter the kinetics of a specific step within the enzyme-reaction cycle in accord with the radical-pair mechanism, although the formulism is not restricted to this specific example. Results show that inclusion of cooperative steps within the enzyme-reaction cycle provides a theoretical basis that enables a simple description of a biphasic response behavior to EMF exposure.

Mechanical vibration in "double-wound" magnetic field exposure coils.

It has been suggested that "double-wound" (bifilar) exposure coils are capable of producing a sham environment in which hum and vibration will be "similar" to the field-exposed condition. We found by direct measurements in a bifilar coil system that vibration amplitude in sham and exposed conditions differed by a factor of 50 when our test system was driven at B = 10 mT. We also found that the normal laboratory environment can include vibrations of an intensity similar to that produced by the exposure system, although not necessarily of similar spectral distribution.

Mechanisms of electromagnetic interaction with cellular systems.

The question of how electromagnetic fields--static or low to high frequency--interact with biological systems is of great interest. The current discussion among biologists, chemists, and physicists emphasizes aspects of experimental verification and of defining microscopic and macroscopic mechanisms. Both aspects are reviewed here. We emphasize that in certain situations nonthermal interactions of electromagnetic fields occur with cellular systems.

Pulsed magnetic field effects on calcium signaling in lymphocytes: dependence on cell status and field intensity.

The effect of 3-Hz, monopolar, quasi-rectangular magnetic field pulses on 45Ca2+ uptake in resting and mitogen-treated rat thymic lymphocytes was evaluated. A 30-min, non-thermal exposure to the pulsed magnetic field (Bpeak = 6.5 mT, Emax = 0.69 mV/cm, Jmax = 2.6 microA/cm2) reduced Concanavalin A-induced 45Ca2+ uptake by 45%. It was observed that (i) the induction of the 3-Hz field response depended on Ca2+ signal transduction activation; (ii) the response direction (stimulation or inhibition) depended on the level of lymphocyte mitogen responsiveness, and (iii) the field response magnitude increased with increasing magnetic field flux densities (Bpeak = 0, 1.6, 6.5 and 28 mT). Our results demonstrate field effects at Bmax nearly 10(4) greater than that of the average human environment for low-frequency magnetic fields and they are consistent with the independent results from other 3-Hz pulsed magnetic field studies with lymphocytes.

Electromagnetic field effects on cells of the immune system: the role of calcium signaling.

During the past decade considerable evidence has accumulated demonstrating that nonthermal exposures of cells of the immune system to extremely low-frequency (ELF) electromagnetic fields (< 300 Hz) can elicit cellular changes that might be relevant to in vivo immune activity. A similar responsiveness to nonionizing electromagnetic energy in this frequency range has also been documented for tissues of the neuroendocrine and musculoskeletal system. However, knowledge about the underlying biological mechanisms by which such fields can induce cellular changes is still very limited. It is generally believed that the cell membrane and Ca(2+)-regulated activity is involved in bioactive ELF field coupling to living systems. This article begins with a short review of the current state of knowledge concerning the effects of nonthermal levels of ELF electromagnetic fields on the biochemistry and activity of immune cells and then closely examines new results that suggest a role for Ca2+ in the induction of these cellular field effects. Based on these findings it is proposed that membrane-mediated Ca2+ signaling processes are involved in the mediation of field effects on the immune system.

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation.

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.

Three-dimensional localization of the NH2- and carboxyl-terminal domain of ribosomal protein S1 on the surface of the 30 S subunit from Escherichia coli.

Antibodies were raised against Escherichia coli ribosomal protein S1 and its NH2- and COOH-terminal fragments, and their specificity was demonstrated by a variety of immunological techniques. These antibodies were then used to investigate the location of protein S1 and its NH2- and COOH-terminal domains on the surface of the 30 S ribosomal subunit by immunoelectron microscopy. In order to prevent dissociation of the protein during the experiments, S1 was cross-linked to 30 S subunits with dithiobis(succinimidyl-propionate); cross-linking yield was 100%. Epitopes of the NH2-terminal domain of S1 were localized at the large lobe of the 30 S ribosomal subunit, close to the one-third/two-thirds partition on the side which in the 70 S ribosome faces the cytoplasm. Experiments with monovalent Fab fragments specific for the COOH-terminal part of S1 provide evidence that the COOH-terminal domain forms an elongated structure extending at least 10 nm from the large lobe of the small subunit into the cytoplasmic space.

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli.

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.

Immunoblotting analysis of protein-protein crosslinks within the 50S ribosomal subunit of Escherichia coli. A study using dimethylsuberimidate as crosslinking reagent.

50S ribosomal subunits of Escherichia coli have been crosslinked with the bifunctional imidoester dimethyl-suberimidate and the protein-protein crosslinks have been analyzed by immunoblotting, using antisera specific for the individual ribosomal proteins of the large ribosomal subunit. Crosslinked protein pairs which occurred in yields higher than 5% have been unambiguously identified. Thus 13 crosslinks have been identified, namely L1-L33, L5-L7/12, L6-L19, L7/12-L10, L7/12-L11, L9-L28, L10-L11, L13-L20, L16-L27, L17-L32, L18-L22, L19-L25 and L27-L33. These data, together with the results which we will be presenting elsewhere, contribute considerably to our knowledge of the protein topography of the 50S ribosomal proteins as determined by immunoelectron microscopy. We can now propose the approximate locations of ten proteins that have not previously been localized.

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques.

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.

A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome.

A three-dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics. The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope-epitope distances on the surface of the large ribosomal subunit with in situ protein-protein cross-linking data. These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model. The model shows (i) that the lower third of the large subunit is protein-poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self-assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the 'back' of the subunit.