Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.

A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr178-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr328-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr178-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr328-) phosphorylation. Interestingly, MST3(Thr328-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr178- /Thr328-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr328-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

Feedback regulation by Atf3 in the endothelin-1-responsive transcriptome of cardiomyocytes: Egr1 is a principal Atf3 target

Endothelin-1 promotes cardiomyocyte hypertrophy by inducing changes in gene expression. Immediate early genes including activating transcription factor 3 (Atf3), Egr1 and Ptgs2 are rapidly and transiently upregulated by endothelin-1 in cardiomyocytes. Atf3 regulates expression of downstream genes and is implicated in negative feedback regulation of other immediate early genes. To identify Atf3-regulated genes, we knocked down Atf3 expression in cardiomyocytes exposed to endothelin-1 and used microarrays to interrogate the transcriptomic effects. Of upregulated mRNAs, expression of 23 (including Egr1, Ptgs2) was enhanced and expression of 25 was inhibited by Atf3 knockdown. Using quantitative PCR, we determined that knockdown of Atf3 had little effect on upregulation of Egr1 mRNA over 30 min, but abolished the subsequent decline, causing sustained Egr1 mRNA expression and enhanced protein expression. This resulted from direct binding of Atf3 to the Egr1 promoter. Mathematical modelling established that Atf3 can suffice to suppress Egr1 expression. Given the widespread co-regulation of Atf3 with Egr1, we suggest that the Atf3-Egr1 negative feedback loop is of general significance. Loss of Atf3 caused abnormal cardiomyocyte growth, presumably resulting from dysregulation of target genes. Our data therefore identify Atf3 as a nexus in cardiomyocyte hypertrophy required to facilitate the full and proper growth response.

Modelling negative feedback networks for activating transcription factor 3 predicts a dominant role for miRNAs in immediate early gene regulation

Activating transcription factor 3 (Atf3) is rapidly and transiently upregulated in numerous systems, and is associated with various disease states. Atf3 is required for negative feedback regulation of other genes, but is itself subject to negative feedback regulation possibly by autorepression. In cardiomyocytes, Atf3 and Egr1 mRNAs are upregulated via ERK1/2 signalling and Atf3 suppresses Egr1 expression. We previously developed a mathematical model for the Atf3-Egr1 system. Here, we adjusted and extended the model to explore mechanisms of Atf3 feedback regulation. Introduction of an autorepressive loop for Atf3 tuned down its expression and inhibition of Egr1 was lost, demonstrating that negative feedback regulation of Atf3 by Atf3 itself is implausible in this context. Experimentally, signals downstream from ERK1/2 suppress Atf3 expression. Mathematical modelling indicated that this cannot occur by phosphorylation of pre-existing inhibitory transcriptional regulators because the time delay is too short. De novo synthesis of an inhibitory transcription factor (ITF) with a high affinity for the Atf3 promoter could suppress Atf3 expression, but (as with the Atf3 autorepression loop) inhibition of Egr1 was lost. Developing the model to include newly-synthesised miRNAs very efficiently terminated Atf3 protein expression and, with a 4-fold increase in the rate of degradation of mRNA from the mRNA/miRNA complex, profiles for Atf3 mRNA, Atf3 protein and Egr1 mRNA approximated to the experimental data. Combining the ITF model with that of the miRNA did not improve the profiles suggesting that miRNAs are likely to play a dominant role in switching off Atf3 expression post-induction.

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

Regulation of ventilation in the caiman (Caiman latirostris): effects of inspired CO2 on pulmonary and upper airway chemoreceptors

In order to study the relative roles of receptors in the upper airways, lungs and systemic circulation in modulating the ventilatory response of caiman (Caiman latirostris) to inhaled CO2, gas mixtures of varying concentrations of CO2 Were administered to animals breathing through an intact respiratory system, via a tracheal cannula by-passing the upper airways (before and after vagotomy), or via a cannula delivering gas to the upper airways alone. While increasing levels of hypercarbia led to a progressive increase in tidal volume in animals with intact respiratory systems (Series 1), breathing frequency did not change until the CO2 level reached 7%, at which time it decreased. Despite this, at the higher levels of hypercarbia, the net effect was a large and progressive increase in total ventilation. There were no associated changes in heart rate or arterial blood pressure. on return to air, there was an immediate change in breathing pattern; breathing frequency increased above air-breathing values, roughly to the same maximum level regardless of the level of CO2 the animal had been previously breathing, and tidal volume returned rapidly toward resting (baseline) values. Total ventilation slowly returned to air breathing values. Administration of CO2 via different routes indicated that inhaled CO2 acted at both upper airway and pulmonary CO2-sensitive receptors to modify breathing pattern without inhibiting breathing overall. Our data suggest that in caiman, high levels of inspired CO2 promote slow, deep breathing. This will decrease deadspace ventilation and may reduce stratification in the saccular portions of the lung.

Phase-Locked loops lock-in range in Frequency Modulated-Atomic Force Microscope nonlinear control system

Since the mid 1980s the Atomic Force Microscope is one the most powerful tools to perform surface investigation, and since 1995 Non-Contact AFM achieved true atomic resolution. The Frequency-Modulated Atomic Force Microscope (FM-AFM) operates in the dynamic mode, which means that the control system of the FM-AFM must force the micro-cantilever to oscillate with constant amplitude and frequency. However, tip-sample interaction forces cause modulations in the microcantilever motion. A Phase-Locked loop (PLL) is used to demodulate the tip-sample interaction forces from the microcantilever motion. The demodulated signal is used as the feedback signal to the control system, and to generate both topographic and dissipation images. As a consequence, a proper design of the PLL is vital to the FM-AFM performance. In this work, using bifurcation analysis, the lock-in range of the PLL is determined as a function of the frequency shift (Q) of the microcantilever and of the other design parameters, providing a technique to properly design the PLL in the FM-AFM system. (C) 2011 Elsevier B.V. All rights reserved.

An approach of optimal sensitivity applied in the tertiary loop of the automatic generation control

This paper proposes an approach of optimal sensitivity applied in the tertiary loop of the automatic generation control. The approach is based on the theorem of non-linear perturbation. From an optimal operation point obtained by an optimal power flow a new optimal operation point is directly determined after a perturbation, i.e., without the necessity of an iterative process. This new optimal operation point satisfies the constraints of the problem for small perturbation in the loads. The participation factors and the voltage set point of the automatic voltage regulators (AVR) of the generators are determined by the technique of optimal sensitivity, considering the effects of the active power losses minimization and the network constraints. The participation factors and voltage set point of the generators are supplied directly to a computational program of dynamic simulation of the automatic generation control, named by power sensitivity mode. Test results are presented to show the good performance of this approach. (C) 2008 Elsevier B.V. All rights reserved.

Frequency-adjustable positive sequence detector for power conditioning applications

This paper proposes a novel and simple positive sequence detector (PSD), which is inherently self-adjustable to fundamental frequency deviations by means of a software-based PLL (Phase Locked Loop). Since the proposed positive sequence detector is not based on Fortescue's classical decomposition and no special input filtering is needed, its dynamic response may be as fast as one fundamental cycle. The digital PLL ensures that the positive sequence components can be calculated even under distorted waveform conditions and fundamental frequency deviations. For the purpose of validating the proposed models, the positive sequence detector has been implemented in a PC-based Power Quality Monitor and experimental results illustrate its good performance. The PSD algorithm has also been evaluated in the control loop of a Series Active Filter and simulation results demonstrate its effectiveness in a closed-loop system. Moreover, considering single-phase applications, this paper also proposes a general single-phase PLL and a Fundamental Wave Detector (FWD) immune to frequency variations and waveform distortions. © 2005 IEEE.

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production. Copyright by the Brazilian Society of Genetics.

Power flow optimization for grid connected inverter using evolutionary algorithm and additional control loop

In this work it is proposed to validate an evolutionary tuning algorithm in plants composed by a grid connected inverter. The optimization aims the tuning of the slopes of P-Ω and Q-V curves so that the system is stable, damped and minimum settling time. Simulation and experimental results are presented to prove the feasibility of the proposed approach. However, experimental results demonstrate a compromising effect of grid frequency oscillations in the active power transferring. In addition, it was proposed an additional loop to compensate this effect ensuring a constant active power flow. © 2011 IEEE.

The effects of different styles of musical auditory stimulation on cardiac autonomic regulation in healthy women

The literature investigated the effects of chronic baroque music auditory stimulation on the cardiovascular system. However, it lacks in the literature the acute effects of different styles of music on cardiac autonomic regulation. To evaluate the acute effects of baroque and heavy metal music on heart rate variability (HRV) in women. The study was performed in 21 healthy women between 18 and 30 years old. We excluded persons with previous experience with music instrument and those who had affinity with the song styles. All procedures were performed in the same sound-proof room. We analyzed HRV in the time (standard deviation of normal-to-normal respiratory rate (RR) intervals, root-mean square of differences between adjacent normal RR intervals in a time interval, and the percentage of adjacent RR intervals with a difference of duration greater than 50 ms) and frequency (low frequency [LF], high frequency [HF], and LF/HF ratio) domains. HRV was recorded at rest for 10 min. Subsequently they were exposed to baroque or heavy metal music for 5 min through an earphone. After the first music exposure they remained at rest for more 5 min and them they were exposed again to baroque or heavy metal music. The sequence of songs was randomized for each individual. The power analysis provided a minimal number of 18 subjects. Shapiro-Wilk to verify normality of data and analysis of variance for repeated measures followed by the Bonferroni test for parametric variables and Friedman's followed by the Dunn's post-test for non-parametric distributions. During the analysis of the time-domain indices were not changed. In the frequency-domain analysis, the LF in absolute units was reduced during the heavy metal music stimulation compared to control. Acute exposure to heavy metal music affected the sympathetic activity in healthy women.

Effects of an isotonic beverage on autonomic regulation during and after exercise

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cardiac autonomic regulation during exposure to auditory stimulation with classical baroque or heavy metal music of different intensities

Objectives: The effects of chronic music auditory stimulation on the cardiovascular system have been investigated in the literature. However, data regarding the acute effects of different styles of music on cardiac autonomic regulation are lacking. The literature has indicated that auditory stimulation with white noise above 50 dB induces cardiac responses. We aimed to evaluate the acute effects of classical baroque and heavy metal music of different intensities on cardiac autonomic regulation. Study design: The study was performed in 16 healthy men aged 18-25 years. All procedures were performed in the same soundproof room. We analyzed heart rate variability (HRV) in time (standard deviation of normal-to-normal R-R intervals [SDNN], root-mean square of differences [RMSSD] and percentage of adjacent NN intervals with a difference of duration greater than 50 ms [pNN50]) and frequency (low frequency [LF], high frequency [HF] and LF/HF ratio) domains. HRV was recorded at rest for 10 minutes. Subsequently, the volunteers were exposed to one of the two musical styles (classical baroque or heavy metal music) for five minutes through an earphone, followed by a five-minute period of rest, and then they were exposed to the other style for another five minutes. The subjects were exposed to three equivalent sound levels (60- 70dB, 70-80dB and 80-90dB). The sequence of songs was randomized for each individual. Results: Auditory stimulation with heavy metal music did not influence HRV indices in the time and frequency domains in the three equivalent sound level ranges. The same was observed with classical baroque musical auditory stimulation with the three equivalent sound level ranges. Conclusion: Musical auditory stimulation of different intensities did not influence cardiac autonomic regulation in men.

Acute auditory stimulation with different styles of music influences cardiac autonomic regulation in men

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)