The calcium-free form of atorvastatin inhibits amyloid-ß(1-42) aggregation in vitro.

Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aß) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aß. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale-Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aß42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aß42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO3-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aß by at least a factor of 2. The 1H-15N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the 16KLVF19 binding site on the Aß peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aß from an α-helix-dominant to a ß-sheet-dominant structure.

Glucose Biosensor Based on Glucose Oxidase Immobilized on BSA Cross-Linked Nanocomposite Modified Glassy Carbon Electrode.

Glucose sensors based blood glucose detection are of great significance for the diagnosis and treatment of diabetes because diabetes has aroused wide concern in the world. In this study, bovine serum albumin (BSA) was used to cross-link glucose oxidase (GOD) on a glassy carbon electrode (GCE) modified by a composite of hydroxy fullerene (HFs) and multi-walled carbon nanotubes (MWCNTs) and protected with a glutaraldehyde (GLA)/Nafion (NF) composite membrane to prepare a novel glucose biosensor. The modified materials were analyzed by UV-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), and cyclic voltammetry (CV). The prepared MWCNTs-HFs composite has excellent conductivity, the addition of BSA regulates MWCNTs-HFs hydrophobicity and biocompatibility, and better immobilizes GOD on MWCNTs-HFs. MWCNTs-BSA-HFs plays a synergistic role in the electrochemical response to glucose. The biosensor shows high sensitivity (167 µA·mM-1·cm-2), wide calibration range (0.01-3.5 mM), and low detection limit (17 µM). The apparent Michaelis-Menten constant Kmapp is 119 µM. Additionally, the proposed biosensor has good selectivity and excellent storage stability (120 days). The practicability of the biosensor was evaluated in real plasma samples, and the recovery rate was satisfactory.

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds.

The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound), and their simultaneous application on the structural and functional features of human αA-crystallin (αA-Cry) using various spectroscopy techniques. Additionally, the surface tension and oligomer size distribution of the treated and untreated protein were tested using tensiometric analysis and dynamic light scattering, respectively. Our results indicated that the reaction of PON and MGO with human αA-Cry leads to the formation of new chromophores, alterations in the secondary to quaternary protein structure, reduction in the size of protein oligomers, and significant enhancement in the chaperone activity of αA-Cry. To reverse the effects of the tested compounds, ascorbic acid and glutathione (main components of lens antioxidant defense system) were applied. As expected, the two antioxidant compounds significantly prevented formation of high molecular weight aggregates of αA-Cry (according to SDS-PAGE). Our results suggest that the lens antioxidant defense system, in particular, glutathione, may provide a strong protection against rapid incidence and progression of diabetic cataract by preventing the destructive reactions of highly reactive DM-associated metabolites.

The Potential Role of Curcumin in Modulating the Master Antioxidant Pathway in Diabetic Hypoxia-Induced Complications.

Oxidative stress is the leading player in the onset and development of various diseases. The Keap1-Nrf2 pathway is a pivotal antioxidant system that preserves the cells' redox balance. It decreases inflammation in which the nuclear trans-localization of Nrf2 as a transcription factor promotes various antioxidant responses in cells. Through some other directions and regulatory proteins, this pathway plays a fundamental role in preventing several diseases and reducing their complications. Regulation of the Nrf2 pathway occurs on transcriptional and post-transcriptional levels, and these regulations play a significant role in its activity. There is a subtle correlation between the Nrf2 pathway and the pivotal signaling pathways, including PI3 kinase/AKT/mTOR, NF-κB and HIF-1 factors. This demonstrates its role in the development of various diseases. Curcumin is a yellow polyphenolic compound from Curcuma longa with multiple bioactivities, including antioxidant, anti-inflammatory, anti-tumor, and anti-viral activities. Since hyperglycemia and increased reactive oxygen species (ROS) are the leading causes of common diabetic complications, reducing the generation of ROS can be a fundamental approach to dealing with these complications. Curcumin can be considered a potential treatment option by creating an efficient therapeutic to counteract ROS and reduce its detrimental effects. This review discusses Nrf2 pathway regulation at different levels and its correlation with other important pathways and proteins in the cell involved in the progression of diabetic complications and targeting these pathways by curcumin.

IAMPE: NMR-Assisted Computational Prediction of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are at the focus of attention due to their therapeutic importance and developing computational tools for the identification of efficient antibiotics from the primary structure. Here, we utilized the 13CNMR spectral of amino acids and clustered them into various groups. These clusters were used to build feature vectors for the AMP sequences based on the composition, transition, and distribution of cluster members. These features, along with the physicochemical properties of AMPs were exploited to learn computational models to predict active AMPs solely from their sequences. Naïve Bayes (NB), k-nearest neighbors (KNN), support-vector machine (SVM), random forest (RF), and eXtreme Gradient Boosting (XGBoost) were employed to build the classification system using the collected AMP datasets from the CAMP, LAMP, ADAM, and AntiBP databases. Our results were validated and compared with the CAMP and ADAM prediction systems and indicated that the synergistic combination of the 13CNMR features with the physicochemical descriptors enables the proposed ensemble mechanism to improve the prediction performance of active AMP sequences. Our web-based AMP prediction platform, IAMPE, is available at http://cbb1.ut.ac.ir/.

Modulating Insulin Fibrillation Using Engineered B-Chains with Mutated C-Termini.

Stress-induced unfolding and fibrillation of insulin represent serious medical and biotechnological problems. Despite many attempts to elucidate the molecular mechanisms of insulin fibrillation, there is no general agreement on how this process takes place. Several previous studies suggested the importance of the C-terminal region of B-chain in this pathway. Therefore, we generated the T30R and K29R/T30R mutants of insulin B-chain. Recombinantly produced wild-type A-chain and mutant B-chains were combined efficiently in the presence of chaperone αB-crystallin. The mutant B-chains along with the control wild-type insulin were used in a wide range of parallel experiments to compare their fibrillation kinetics, morphology of fibrils, and forces driving the fibril formation. The mutant insulins and their B-chains displayed significant resistance against stress-induced fibrillation, particularly at the nucleation stage, suggesting that the B-chain might be influencing the insulin fibrillation. The fact that the different mature insulins formed larger fibrillar bundles compared to those formed by their B-chains alone suggested the role of A-chain in the lateral association of the insulin fibrils. Overall, in addition to the N-terminal region of the B-chain, which was shown to serve as an important regulator of insulin fibrillation, the C-terminal region of this peptide is also crucial for the control of fibrillation, likely serving as an attachment site engaged in the formation of the nucleus and protofibril. Finally, two mutated insulin variants examined in this study might be of interest to the pharmaceutical sector as, to our knowledge, novel intermediate-acting insulin analogs because of their suitable biological activity and improved stability against stress-induced fibrillation.

Personalizing the safe, appropriate and effective concentration(s) of ozone for a non-diabetic individual and four type II diabetic patients in autohemotherapy through blood hemoglobin analysis.

BACKGROUND: Diabetes is a chronic disease associated with many problems and high costs. In recent decades, a lot of research has been carried out in order to improve methods of treatment of diabetic patients. One of the currently used complementary therapies for diabetes is ozone therapy or autohemotherapy. The beneficial effects of ozone has been proven in many diseases such as diabetes, but the critical issue is the determination of the safe and effective concentration of ozone reacting with blood and in particular hemoglobin. METHODS: A number of spectroscopic techniques including intrinsic fluorescence, circular dichroism and UV-VIS spectroscopies were used as well as SDS-PAGE, Native-PAGE and dynamic light scattering to analyze the effect of ozonation on hemoglobin of a non-diabetic individual and four diabetic patients in order to find the appropriate concentration(s) of ozone for personalized autohemotherapy. RESULTS: In this study, we determined the personalized concentration(s) for a safe and effective ozonation of a non-diabetic individual and four diabetic type II patients, based on blood hemoglobin analysis. CONCLUSIONS: A number of techniques were used to determine the personalized ozone concentration(s) for a safe and effective autohemotherapy based on blood hemoglobin analysis. SDS-PAGE and dynamic light scattering were identified as the two main techniques needed for personalizing the ozone concentration(s) for each individual as otherwise hemoglobin in blood can oligomerise and cause serious damage if the inappropriate ozone concentration is used.

A first-passage approach to the thermal breakage of a discrete one-dimensional chain.

Using the first passage method for a Markov process, we theoretically study the fragmentation rate of a discrete one-dimensional chain (Rouse model). The fragmentation occurs due to thermal fluctuations. Assuming equilibrium initial conditions, we obtain an expression for the fragmentation rate of the one-dimensional filament as a function of the number of monomers, the position of the breaking point along the filament, the ratio of the bond energy to the thermal energy, and the Rouse relaxation time. We also obtain the fragmentation rate for a fixed initial configuration of the chain by numerically solving a Volterra equation. Our results reduce to those of previous theoretical studies at the appropriate limits, and spell out the role of the relevant time scales. The prediction of our model for the fragmentation rate of insulin fibrils under optimal growth conditions for the solution appears to be consistent with experimental data from other studies.

Activation of catalase by pioglitazone: Multiple spectroscopic methods combined with molecular docking studies.

Pioglitazone is an important prescription antidiabetic drug with positive roles in controlling high blood sugar in patients with type 2 diabetes. In the present study, we investigated the effects of pioglitazone on the structure and function of bovine liver catalase (BLC) using different spectroscopic and theoretical methods. UV-Vis absorption, fluorescence spectroscopy, synchronous fluorescence, and circular dichroism studies revealed conformational changes in the BLC structure and heme group in the presence of different concentrations of pioglitazone. Kinetic studies indicated that pioglitazone can increase BLC activity by approximately threefold compared with free enzyme. The fluorescence quenching data showed one binding site for pioglitazone, and the binding constants at 298, 304, and 310 K were calculated as 5.01 × 107  M-1 , 5.8 × 107  M-1 , and 6.6 × 107  M-1 , respectively. The static type of quenching mechanism was mainly involved in the quenching of intrinsic emission of the enzyme. Thermodynamic data suggested that hydrophobic interactions played a major role in the binding reaction of pioglitazone with BLC. The molecular docking studies indicated that pioglitazone interacts with the cavity in the middle of the ß-barrel and wrapping domain of BLC. Thus, pioglitazone can increase catalase activity by changing the BLC structure.

The impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C.

A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. While Arg54 is highly conserved within different species, the cataractogenic mutations R54L, R54P and R54C have been recently identified in CRYAA gene, encoding human αA-Cry. The detailed structural and functional aspects, stability and amyloidogenic properties of αA-Cry were determined upon the above-mentioned missense mutations, using various spectroscopic techniques, gel electrophoresis, electron microscopy, size exclusion chromatography analyses, and chaperone-like activity assay. The different mutations at Arg54 result in diverse structural alterations among mutant proteins. In addition, the mutant proteins displayed reduced thermal stability, increased amyloidogenic properties and attenuated chaperone-like activity against aggregation of γ-Cry, catalase and lysozyme. The mutant proteins were also capable of forming larger oligomeric complexes with γ-Cry which is the natural partner of α-Cry in the eye lenses. The most significant structural and functional damages were observed upon R54L mutation which was also accompanied with increased oligomeric size distribution of the mutant protein. The cataractogenic nature of R54P mutation can be explained with its detrimental effect on chaperone-like activity, conformational stability and proteolytic digestibility of the mutant protein. Also, R54C αA-Cry displayed an important intrinsic propensity for disulfide protein cross-linking with significantly reduced chaperone-like activity against all client proteins. These mutations revealed a range of detrimental effects on the structure, stability and functional properties of αA-Cry which all together can explain the pathomechanisms underlying development of the associated congenital cataract disorders.

Protective role of antioxidant compounds against peroxynitrite-mediated modification of R54C mutant αA-crystallin.

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has endogenous production in the eye ball and contributes to a variety of ocular disorders. In the current study the structural characteristics, chaperone-like activity and conformational stability of R54C mutant αA-crystallin (αA-Cry) were studied upon modification with PON and in the presence of three antioxidant compounds such as ascorbic acid (ASA), glutathione (GSH) and N-acetylcysteine (NAC) using gel electrophoresis and different spectroscopy methods. The results of both fluorescence analysis and gel electrophoresis suggested that PON modification leads to dityrosine-mediated intermolecular cross-linking of this cataractogenic mutant protein. Also, the propensity of R54C mutant αA-Cry for disulfide cross-linking was increased upon PON modification. In addition, the PON-modified protein indicated structural alteration, reduced chemical stability and different pattern of proteolysis. Upon modification with PON, mutant αA-Cry displayed a significant increase in the chaperone-like activity against aggregation of γ-crystallin and insulin. In addition, different antioxidant compounds indicated a prominent role in neutralizing the PON damaging effects on structural integrity and stability of this protein. The results of this study may highlight the importance of antioxidant-rich foods or potent antioxidant supplements in protection of lens crystallins against PON-mediated structural damages and cataract development.

Destructive effect of non-enzymatic glycation on catalase and remediation via curcumin.

Non-enzymatic glycation of proteins is a post-translational modification that is produced by a covalent binding between reducing sugars and amino groups of lysine and arginine residues. In this paper the effect of pathological conditions, derived from hyperglycemia on bovine liver catalase (BLC) as a model protein was considered by measuring enzyme activity, reactive oxygen species (ROS) generation, and changes in catalase conformational properties. We observed that in the presence of glucose, the catalase activity gradually decreased. ROS generation was also involved in the glycation process. Thus, decreased BLC activity was partly considered as a result of ROS generation through glycation. However, in the presence of curcumin the amount of ROS was reduced resulting in increased activity of the glycated catalase. The effect of high glucose level and the potential inhibitory effect of curcumin on aggregation and structural changes of catalase were also investigated. Molecular dynamic simulations also showed that interaction of catalase with curcumin resulted in changes in accessible surface area (ASA) and pKa, two effective parameters of glycation, in potential glycation lysine residues. Thus, the decrease in ASA and increase in pKa of important lysine residues were considered as predominant factors in decreased glycation of BLC by curcumin.

Red/ox states of human protein disulfide isomerase regulate binding affinity of 17 beta-estradiol.

Human protein disulfide isomerase (hPDI) is a key redox-regulated thiol-containing protein operating as both oxidoreductase and molecular chaperone in the endoplasmic reticulum of cells. hPDI thiol-disulfide interchange reactions lead to the adoption of two distinct red/ox conformations with different substrate preferences. hPDI also displays high binding capacity for some endogenous steroid hormones including 17 beta-estradiol (E2) and thus contributes to the regulation of their intracellular concentration, storage and actions. The primary focus of this study was to investigate the impact of E2 binding on functional activity of recombinant hPDI. Then, we examined the effect of E2 binding on structural alteration of hPDI red/ox conformations and its influence on affinity and position of interaction using experimental and computational analysis. Our results revealed that interaction of one E2 per each hPDI molecule led to the inhibition of hPDI reductase activity and conformational changes in both oxidation states. Mutually, E2-binding position were also redox-regulated with higher affinity in oxidized hPDI compare to the reduced form. The importance of histidine-256 protonation states in distinct binding preferences of E2 were also demonstrated in hPDI red/ox conformations. These findings might pave the way for better understanding of the mechanisms behind the redox-dependent hormone-binding activity of hPDI.

Histidine substitution in the most flexible fragments of firefly luciferase modifies its thermal stability.

Molecular dynamics (MD) at two temperatures of 300 and 340 K identified two histidine residues, His461 and His489, in the most flexible regions of firefly luciferase, a light emitting enzyme. We therefore designed four protein mutants H461D, H489K, H489D and H489M to investigate their enzyme kinetic and thermodynamic stability changes. Substitution of His461 by aspartate (H461D) decreased ATP binding affinity, reduced the melting temperature of protein by around 25 °C and shifted its optimum temperature of activity to 10 °C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Similar changes were observed for H489K and, to a lesser degree, H489M mutations. Based on our results we conclude that the MD simulation-based rational substitution of histidines by salt-bridge forming residues can modulate conformational dynamics in luciferase and shift its optimal temperature activity.

Detection of Guanine and Adenine Using an Aminated Reduced Graphene Oxide Functional Membrane-Modified Glassy Carbon Electrode.

A new electrochemical sensor based on a Nafion, aminated reduced graphene oxide and chitosan functional membrane-modified glassy carbon electrode was proposed for the simultaneous detection of adenine and guanine. Fourier transform-infrared spectrometry (FTIR), transmission electron microscopy (TEM), and electrochemical methods were utilized for the additional characterization of the membrane materials. The prepared electrode was utilized for the detection of guanine (G) and adenine (A). The anodic peak currents to G and A were linear in the concentrations ranging from 0.1 to 120 µM and 0.2 to 110 µM, respectively. The detection limits were found to be 0.1 µM and 0.2 µM, respectively. Moreover, the modified electrode could also be used to determine G and A in calf thymus DNA.

Appraisal of role of the polyanionic inducer length on amyloid formation by 412-residue 1N4R Tau protein: A comparative study.

In many neurodegenerative diseases, formation of protein fibrillar aggregates has been observed as a major pathological change. Neurofibrillary tangles, mainly composed of fibrils formed by the microtubule-associated protein; Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer's disease. Tau belongs to the class of natively unfolded proteins and partially folds into an ordered ß-structure during aggregation. Polyanionic cofactors such as heparin are commonly used as inducer of Tau aggregation in vitro. The role of heparin in nucleation and elongation steps during Tau fibril formation is not fully understood. In the current study, aggregation kinetics as well as structure of Tau amyloid fibrils, by using the 1N4R isoform, have been reproducibly determined in the presence of heparin and the shorter molecule; enoxaparin. The kinetic studies demonstrated that heparin (not enoxaparin) efficiently accelerates Tau amyloid formation and revealed, mechanistically, that the molecular weight of the inducer is important in accelerating amyloidogenesis. The kinetic parameter values of Tau amyloid aggregation, especially, the amyloid aggregation extent, were relatively different in the presence of heparin and enoxaparin, at various stoichiometries of the inducers binding. Also, based on the results, obtained from CD, FTIR, AFM and XRD studies, it may be suggested that the inducer length plays a critical role mainly in the nucleation process, so that it determines that oligomers lie on or off the pathway of Tau fibrillization. The biochemical results herein suggest that the chemical environment of the extracellular matrix as well as localization of distinct glycosaminoglycans may influence deposition behavior of Tau amyloidosis.

Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact.

Quantitative sequence-activity modeling of antimicrobial hexapeptides using a segmented principal component strategy: an approach to describe and predict activities of peptide drugs containing L/D and unnatural residues.

The treatment of infections caused by multi-drugs resistant bacteria and fungi is a particular challenge. Whereas cationic antimicrobial peptides (CAPs) are considered as promising drug candidates for treatment of such superbugs, recent studies have focused on design of those peptides with increased bioavailability and stability against proteases. In between, applications of the quantitative structure-activity relationship (QSAR) studies which provide information on activities of CAPs based on descriptors for each individual amino acid are inevitable. However, the satisfactory results derived from a QSAR model depend highly on the choice of amino acid descriptors and the mathematical strategy used to relate the descriptors to the CAPs' activity. In this study, the quantitative sequence-activity modeling (QSAM) of 60 CAPs derived from O-W-F-I-F-H(1-Bzl)-NH2 sequence which showed excellent activities against a broad range of hazardous microorganisms: e.g., MRSA, MRSE, E. coli and C. albicans, is discussed. The peptides contained natural and non-natural amino acids (AAs) of the both isomers D and L. In this study, a segmented principal component strategy was performed on the structural descriptors of AAs to extract AA's indices. Our results showed that constructed models covered more than 82, 94, 80 and 78 % of the cross-validated variance of C. albicans, MRSA, MRSE and E. coli data sets, respectively. The results were also used to determine the important and significant AAs which are important in CAPs activities. According to the best of our knowledge, it is the first successful attempt in the QSAM studies of peptides containing both natural and non-natural AAs of the both L and D isomers.

Effects of 940 MHz EMF on bioluminescence and oxidative response of stable luciferase producing HEK cells.

The effects of mobile phone frequency electromagnetic field (RF-EMF, 940 MHz) on a stable cell line (HEK293T) harbouring the firefly luciferase gene were evaluated. A waveguide exposure system with 1 W input power provided the mean specific absorption rate of ≈0.09 W kg(-1) in 35 mm Petri dishes. The effects of exposure duration (15, 30, 45, 60 and 90 min) on luciferase activity and oxidative response elements were investigated. Endogenous luciferase activity was reduced after 30 and 45 min of continuous exposure, while after 60 min, the exposed cell lysate showed higher luciferase activity compared with the non-exposed control. Reactive oxygen species (ROS) generation was highest in the 30 min exposed cells as studied by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. The observed boost in ROS was then followed by a sharp rise in catalase (CAT) and superoxide dismutase (SOD) activity and elevation of glutathione (GSH) during the 45 min exposure. Decrease in lipid peroxidation (malondialdehyde, MDA) was meaningful for the 45 and 60 min exposed cells. Therefore, it appears that an increase in the activity of luciferase after 60 min of continuous exposure could be associated with a decrease in ROS level caused by activation of the oxidative response. This ability in cells to overcome oxidative stress and compensate the luciferase activity could also be responsible for the adaptive response mechanism detected in ionizing radiation studies with RF-EMF pre-treatments.