Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of capase-3, and increasing cell death. The over-expressaion of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. lit cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitiosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

Role of sigma(54) in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Expression Profile of Rat Hippocampal Neurons Treated with the Neuroprotective Compound 2,4-Dinitrophenol: Up-Regulation of cAMP Signaling Genes

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Optimal regulation of oil fields under asymmetric information

Neste trabalho é analisada a relação entre um regulador e uma empresa petrolífera. Há várias incertezas inerentes à essa relação e o trabalho se concentra nos efeitos da assimetria de informação. Fazemos a caracterização da regulação ótima sob informação assimétrica, quando o regulador deve desenhar um mecanismo que induz a firma a revelar corretamente sua informação privada. No caso em que a rma não pode se comprometer a não romper o acordo, mostramos que o regulador pode não implementar o resultado ótimo que é obtido sob informação completa. Nesse caso, o regulador não consegue compartilhar os riscos com a firma de forma ótima. Por fim, é apresentado um exemplo, em que mostramos que a condição de Spence-Mirrlees (SMC) pode não valer. Esse resultado aparece de forma natural no modelo.

Beyond ‘best practices': the international regulation of capital markets

It is widely acknowledged that there is considerable international pressure for international ‘best practices’ to be adopted via national legislation. This would occur either by means of model laws or through the passing of country specific legislation that closely replicates foreign legal formats, administrative rules, and or regulation. These attempts to spread the implementation of ‘best practices’ have gained importance in the international debate due to the liberalization of international capital flows. The oversight, country reports, and technical assistance carried out by international organizations along with the growing internationalization of investors have also contributed to this growing pressure. In this respect, due to the constant evolution of transactions and the end objective of making sure that capital markets are developed with just rules, structures, and methods, this article looks to analyze the adoption of standardized models of capital market regulation. Furthermore it looks to examine the motivation and interest of states and other ‘stakeholders’ at the international level.

How to evaluate financial regulation of Brazil

The purpose of this paper is to evaluate the current financial regulation of Brazil, with emphasis on the changes that have occurred after the 2008 global crisis. Before that, this introduction presents the analytical perspective that will be used to think over the financial regulation of an emerging country, such as Brazil.

Trade regulation of exchange rates

O presente estudo busca analisar o quadro regulatório internacional referente a medidas cambiais que trazem impactos no comércio. O artigo pretende explorar como a questão do câmbio se relaciona à OMC e afeta seus instrumentos e princípios para, em seguida, buscar dispositivos nos Acordos da OMC que poderiam ser aplicados à questão cambial a fim de reequilibrar os impactos causados pelos desalinhamentos cambiais no comércio internacional

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly switched on by specific proteolytic cleavage.

The adrenergic regulation of the cardiovascular system in the South American rattlesnake, Crotalus durissus

The present study investigates adrenergic regulation of the systemic and pulmonary circulations of the anaesthetised South American rattlesnake, Crotalus durissus. Haemodynamic measurements were made following bolus injections of adrenaline and adrenergic antagonists administered through a systemic arterial catheter. Adrenaline caused a marked systemic vasoconstriction that was abolished by phentolamine, indicating this response was mediated through alpha-adrenergic receptors. Injection of phentolamine gave rise to a pronounced vasodilatation (systemic conductance (G(sys)) more than doubled), while injection of propranolol caused a systemic vasoconstriction, pointing to a potent alpha-adrenergic, and a weaker beta-adrenergic tone in the systemic vasculature of Crotalus. Overall, the pulmonary vasculature was far less responsive to adrenergic stimulation than the systemic circulation. Adrenaline caused a small but non-significant pulmonary vasodilatation and there was tendency of reducing this dilatation after either phentolamine or propranolol. Injection of phentolamine increased pulmonary conductance (G(pul)), while injection of propranolol produced a small pulmonary constriction, indicating that alpha-adrenergic and beta-adrenergic receptors contribute to a basal regulation of the pulmonary vasculature. Our results suggest adrenergic regulation of the systemic vasculature, rather than the pultrionary, may be an important factor in the development of intracardiac shunts. (c) 2007 Elsevier B.V. All rights reserved.

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.

Involvement of median raphe nucleus 5-HT1A receptors in the regulation of generalized anxiety-related defensive behaviours in rats

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

Hymenolepis diminuta: Praziquantel removal of adult tapeworms is followed by apoptotic down-regulation of mucosal mastocytosis

The rat tapeworm, Hymenolepis diminuta, induces mastocytosis, hypertrophy of enteric smooth muscle, alteration of enteric myoelectric activity, and slowed enteric transit of the rat host's intestine. This report examines the resolution of both tapeworm-induced mastocytosis and tissue changes during the period following removal of the tapeworm with Praziquantel (PZQ). The dynamics of the mucosal mast cell (MMC) population following removal of the tapeworms was assessed by histochemical identification of MMC and morphometric techniques. As a possible mechanism of MMC population regulation, MMC apoptosis was examined over the same experimental period using the in situ nick end labeling of fragmented DNA (TUNEL). Shifts in MMC numbers were correlated with functional and morphological changes of the intestine following removal of the adult-stage tapeworm. Ileal tissues from rats infected 32 days with H. diminuta (the beginning of plateau phase of tapeworm-induced chronic mastocytosis) were harvested 1, 2, 3, and 4 weeks after the PZQ treatment. Control ilea were obtained either from rats which were never infected and never treated with PZQ or from rats infected with H, diminuta for 32 days but not treated with PZQ. In order to detect MMC and apoptosis, tissue sections of ileum were doubled stained sequentially with Astra blue for MMC granules followed by a modification of the TUNEL technique. No alteration in MMC numbers were observed in PZQ-treated animals until 3 weeks after the removal of the tapeworms. The decline of MMC occurred in the mucosa and submucosa. MMC numbers first approached uninfected control levels at 4 weeks posttreatment. Coincident with the decline in mucosal MMC numbers, the rate of MMC entering apoptosis also declined. Simultaneously, ileal smooth muscle layers, hypertrophied by infection, and mucosal structures began the process of involution and atrophy. Apoptosis of MMC in the submucosa and muscularis mucosa was not detected. In conclusion, H. diminuta elicited mastocytosis and increased thickness of both mucosa and muscularis externa do not begin a decline toward control Values until 3 weeks after the parasites are gone and normal intestinal motility is restored. These data are consistent with the lack of MMC mediation of altered motility, and the decline in the rate of MMC apoptosis at 3 weeks post-PZQ suggests that apoptosis may play an important role in the involution of tapeworm-induced mastocytosis. (C) 1999 Academic Press.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 +/- 0.48 vs food-restricted group = 4.84 +/- 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 +/- 0.44 vs food-restricted group = 7.96 +/- 0.45, and control = 1.52 +/- 0.06 vs food-restricted group = 1.53 +/- 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.