Effects of 940 Hz EMF on luciferase solution: structure, function, and dielectric studies.

We designed a rectangular waveguide exposure system to study the effects of mobile phone frequency (940 MHz) electromagnetic fields (EMF) on luciferase structure and activity. The luciferase activity of exposed samples was significantly higher than that of unexposed samples. Dynamic light scattering of the exposed samples showed smaller hydrodynamic radii compared to unexposed samples (20 nm vs. 47 nm ± 5%). The exposed samples also showed less tendency to form aggregates, monitored by turbidity measurements at l = 360 nm. A microwave dielectric measurement was performed to study the hydration properties of luciferase solutions with a precision network analyzer over frequency ranges from 0.2 to 20 GHz before and after exposure. The change in the dielectric properties of the exposed luciferase solution was related to the disaggregation potency of the applied field. Together, our results suggested that direct interactions with luciferase molecules and its dipole moment were responsible for the reduced aggregation and enhanced luciferase activity upon exposure to the EMF.

An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate.

The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates: luciferin, ATP and oxygen. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. The dose constant for removal of oxygen is 70 +/- 20 Gy, a much smaller dose than required to cause damage to protein. Removal of oxygen, which is initially in excess, leads to a sigmoidal response of bioluminescence to radiation dose, consistent with a Michaelis-Menten relationship to substrate concentration. When excess oxygen is exhausted, the response becomes exponential. Following the irradiation, bioluminescence recovers due to a slow leak of oxygen into the solution. This may also explain previous observations on the response of bioluminescent bacteria to radiation. We have studied the dependence of the reaction rate on enzyme and substrate concentration and propose a model for the reaction pathway consistent with this data. The light output from unirradiated samples decreases significantly with time due to product inhibition. We observe that this inhibition rate changes dramatically immediately after a sample is exposed to the beam. This sudden change of the inhibition rate is unexplained but shows that enzyme regulatory function responds to ionizing radiation at a dose level less than 0.6 Gy.

Single-cell imaging of c-fos expression in rat primary hippocampal cells using a luminescence microscope.

Methods to study gene expression in live cells over time have been limited. One known method is the luciferase assay, which measures the luminescence of luciferase by coupling its expression to the promoter of a gene under study. This luminescence in cells can be measured over time by a luminometer. One major drawback of the luminometer, however, is that it can only measure the luminescence of a group of cells, and cannot follow the differences that may exist among individual cells. A novel luminescence microscope allows the visualization of individual luminescent cells over time through CCD photography. In this study, live single cells of the rat hippocampus were observed under the microscope for luciferase expression driven by the c-fos promoter. We showed that the cell body and neurite areas within a single neuron exhibited differences in luminescence. Because this microscope could detect differences among subcellular regions of single-cell, it may be a promising novel tool to study polarized cells like neurons, and to elucidate proteins involved in neuronal processes such as dendritic/axonal targeting and synaptogenesis.

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.

Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors.

Light emission from the bacterial luciferase operon has been variously exploited during last two decades. The use of convenient inducible promoters has granted significant degrees of specificity to whole cell-based assays for high-throughput screening and environmental monitoring. Nevertheless, unexplained unspecific responses have been repeatedly reported. Here, we show that the impairment of the intracellular biochemical equilibrium interferes with the luminescence produced by Escherichia coli and Staphylococcus aureus strains carrying the lux operon under constitutive or inducible control. Compounds as trimethoprim and methotrexate, by indirectly inducing NADPH accumulation, enhance light emission. Conversely, molecules driving the cell toward an oxidized state, as dimethyl sulfoxide, inhibit luminescence. These findings fit into the accepted biochemical pathway for bioluminescence, where NADPH and reducing equivalents are necessary for the production of luciferase substrates, although they do not directly take part into the light-emitting reaction. Moreover, we investigated the influence of induction timing upon the bioluminescence response from inducible reporter systems and demonstrated a correlation between the emitted light and the growth phase at which induction is performed. Our results provide explanations for some unspecific responses recorded so far in whole cell-based luminescent biosensors and emphasize the intrinsic limitations of this kind of reporting system.

Spatially discrete, light-driven protein expression.

Transgene-based inducible expression systems offer the potential to study the influence of any gene at any point during an organism's lifetime. However, the expression of individual genes is both temporally and spatially (i.e., cell/tissue)-regulated. The inducible gene expression systems devised to date do not offer fine spatial control over gene expression. We describe herein the creation and study of a light-activatable, ecdysone-inducible gene expression system. We have constructed the first example of a caged ecdysteroid, which is virtually inactive as an inducing agent in a luciferase-based gene expression system. However, upon exposure to brief illumination, the caged ecdysteroid is rapidly converted into active beta-ecdysone. Caged beta-ecdysone is cell permeable, can be intracellularly photouncaged, and, in combination with spot illumination, can be used to drive spatially discrete protein expression in a multicellular setting.

Effects of iodide on the fluorescence and activity of the hydroperoxyflavin intermediate of Vibrio harveyi luciferase.

The 4a-hydroperoxy-4a,5-dihydroFMN intermediate (II or HFOOH) of Vibrio harveyi luciferase is known to transform from a low quantum yield IIx to a high quantum yield (lambdamax 485 nm, uncorrected) IIy fluorescent species on exposure to excitation light. Similar results were observed with II prepared from the alphaH44A luciferase mutant, which is very weak in bioluminescence activity. Because of the rapid decay of the alphaH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product oxidized flavin mononucleotide (FMN). Potassium iodide (KI) at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type (WT) and alphaH44A luciferase readily detectable. For both II species, the luciferase-bound peroxyflavin was well shielded from KI quenching. KI also enhanced the decay rates of both the WT and alphaH44A II. For alphaH44A, the transformation of IIx to IIy can be induced by KI in the dark, and it is proposed to be a consequence of a luciferase conformational change. The WT II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II.

A critical analysis of the use of radiation inactivation to measure the mass of protein.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.

The kinetics of radiation damage to the protein luciferase and recovery of enzyme activity after irradiation.

Experimental observations are reported which follow the bioluminescence intensity of luciferase during irradiation by a 5 MeV proton beam. Bioluminescence is a measure of the protein enzyme activity and provides an assay of the enzyme rate of reaction in real time. Transient responses after a pulse of protons show recovery of the reaction rate with two time constants of 0.3 s(-1) and 0.01 s(-1). Changes in the reaction rate are due to radiation damage to the active form of the protein luciferase. Quantitative analysis of the radiation damage and recovery of the protein shows that products of the radiolysis of water play major part in the process of enzyme damage at room temperature. A few minutes after the pulse of protons, most of the enzyme activity has recovered. We attribute the fast recovery to the removal of charged ions, while the slow recovery involves refolding of denatured protein.

Use of bleomycin- and heat shock-induced calreticulin promoter for construction of a mammalian expression vector.

Addition of bleomycin (Bm) to an NIH/3T3 cell culture induced the overproduction of four cellular proteins [Kumagai and Sugiyama (1998) J. Biochem. 124, 835-841]. The two proteins were identified on N-terminal amino acid sequence analysis as calreticulin and mitochondrial matrix protein P1, which are known as heat shock proteins, respectively. In this study, we cloned the calreticulin promoter region from the genomic DNA of NIH/3T3 cells and observed that heat shock treatment at 42 degrees C or the addition of Bm to the cell culture caused overexpression of the luciferase gene controlled by the cloned calreticulin promoter. This suggests that Bm induces the transcriptional activation of stress-heat shock genes. We constructed an expression vector for mammalian cells, which is controlled by the calreticulin promoter.

Creation of a thermostable firefly luciferase with pH-insensitive luminescent color.

The thermal instability and pH-sensitive spectral property of firefly luciferase have hampered its use as a sensitive multicolor luminescent label or bioluminescent resonance energy transfer donor. With the intention of improving the thermostability of a previously found firefly Hotaria parvula luciferase mutant with minor pH-sensitive spectral change (V368A), further mutation (E356R) was introduced by taking a reportedly stabilized mutant of Photinus pyralis luciferase into account. The double mutant E356R/V368A showed significantly improved thermostability because > 90% activity remained after incubation for 1 h at 45 degrees C, with its specific activity being maintained. Unlike the wild type or V368A, E356R/V368A showed no change in the emission maximum of 568 nm even at pH 6.3, also implying a mutual relationship between thermostability and the proportion of yellow-green luminescent peak under acidic condition.

Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis.

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.