Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.     

NADP pool state and activity of NADP-dependent dehydrogenases in the heart tissue of adult and old rats during immobilization stress

To reveal the reasons of age-specific change of heart sensitivity to stress, the content of oxidized and reduced NADP and activity of NADP-dependent dehydrogenase in myocardium of adult and old immobilized rats have been studied. It has been established that in the process of aging the amount of reduced NADP decreases in the old rat heart. At the same time the activity of NADP-dependent dehydrogenase decreases in postmitochondrial fraction of old rat myocardium as compared to adult rats. There is no change in the amount of reduced NADP in the heart of both rat age groups after 30-minute immobilization. The activity of NADP-dependent dehydrogenase in postmitochondrial fraction of myocardium in adult rats remains on the initial level while activation of NADP-dependent malate dehydrogenase and glucose 6-phosphate dehydrogenase appears in old rats.     

Suppression of gamma EEG activity as an index of a spreading depression wave in the neocortex of a waking rabbit

A spreading depression (SD) can spontaneously develop in seizures, attacks of migraine, vascular disorders and other pathological states of the brain. However, problems in technique of recording the DC-potential in the neocortex of humans and waking animals substantially restrict the possibilities of studying functional consequences of the SD. In this article, the EEG pattern was studied in detail at the moment of the SD development. Specific features were revealed, which make it possible to detect the SD without recording shifts of the DC-potential. At the moment of the SD arrival, the interhemispheric balance drastically disturbs because of a strong decrease in the high-frequency activity. By the time indices, the course of the suppression of the gammal and gamma2 EEG frequencies is the most reliable symptom of the SD wave development. The EEG spectral power in the delta band increases with a certain delay in reference to the deep depression of the high-frequency activity and is, in essence, an SD aftereffect. The found EEG signs of an SD wave can substantially simplify the identification of this phenomenon both in experiment and clinical conditions in certain pathological states of the brain.     

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe

To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

Gender differences in regulating emotions in response to text with violent content

Gender difference in intensity of emotional response depending on the cognitive disclosure of the emotions were analyzed in detail. Texts with emotionally negative content were presented to the subjects, and the parameters of their cardiovascular activity were recorded either before or after filling a special checklist to assess the impression caused by the texts and to reveal changes in the physiological activation of the subjects in response to these texts. Unlike women, men intensified their affective involvement into processing the content of the texts after filling the checklist. The results are interpreted in the context of current research on physiological emotional processes during their cognitive disclosure; the role of gender differences in the regulation of these processes was also studied.     

Decision of mathematical logical tasks in sensory enriched environment (classical music)

The time of a decision of mathematical logical tasks (MLT) was decreased during classical musical accompaniment (power 35 and 65 dB). Music 85 dB did not influence on the process of decision of MLT. Decision without the musical accompaniment led to increasing of coherent function values in beta1, beta2, gamma frequency ranges in EEG of occipital areas with prevalence in a left hemisphere. A coherence of potentials was decreased in EEG of frontal cortex. Music decreasing of making-decision time enhanced left-sided EEG asymmetry The intrahemispheric and the interhemispheric coherences of frontal cortex were increased during the decision of MLT accompanied by music. Using of musical accompaniment 85 dB produced a right-side asymmetry in EEG and formed a focus of coherent connections in EEG of temporal area of a right hemisphere.     

Interhemispheric EEG interrelations in recognition of masked visual images accompanied by music

Changes in the EEG depend on the style of accompanying music. Classical music produces more patterns with increased diagonal coherences in the γ frequency band, where two foci of integration are formed at points T ₄ and T ₆. During listening to rock music, a larger proportion of all changes occurred in the θ-and α₁ frequency bands; an integration focus was formed at point T ₆ in the α₁ and γ frequency bands. An increase in diagonal coherences that was observed during the recognition was especially pronounced in the Δ-, θ-and α₁ frequency bands. In addition, there was an increase in the coherence between the potentials recorded at points O ₁-P ₄ and O ₂-F ₇, and it was observed in all the frequency bands. During the recognition of masked visual images accompanied by classical or rock music, there were more patterns with an increased diagonal coherence of the currently dominant activity. Before erroneous recognition, a widely generalized increase in coherence over the brain cortex was observed in the Δ, θ, and α₁ bands, which was an informative indicator of the subsequent erroneous recognition. In tests with recognition unaccompanied by music, as well as with music, the main change in symmetrical coherences was either a decrease in or an absence of changes in this indicator versus the background value.