Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Synthesis, electrochemical studies and anticancer activity of ferrocenyl oxindoles

A series of (E) and (Z)-ferrocenyl oxindoles were prepared by coupling substituted oxindoles to ferrocenylcarboxyaldehyde in the presence of morpholine as a catalyst. The redox behavior of these isomers was determined by cyclic voltammetry. The effects of the oxindole derivatives on the migration of human breast cancer cells were evaluated using the wound-healing assay and the Boyden chamber cell-migration assay. The most potent Z isomers 11b (IC(50) = 0.89 mu M), 12b (IC(50) = 0.49 mu M) and 17b (IC(50) = 0.64 mu M) could represent attractive new lead compounds for further development for cancer therapy.

Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial K(ATP) Channel Activation

Background: The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. Methodology/Principal Findings: Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca(2+) induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K(+) transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoK(ATP) activation. Conclusions/Significance: We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoK(ATP) activation.

Insulin and leptin relations in obesity: a multimedia approach

Obesity has been recognized as a worldwide public health problem. It significantly increases the chances of developing several diseases, including Type II diabetes. The roles of insulin and leptin in obesity involve reactions that can be better understood when they are presented step by step. The aim of this work was to design software with data from some of the most recent publications on obesity, especially those concerning the roles of insulin and leptin in this metabolic disturbance. The most notable characteristic of this software is the use of animations representing the cellular response together with the presentation of recently discovered mechanisms on the participation of insulin and leptin in processes leading to obesity. The software was field tested in the Biochemistry of Nutrition web-based course. After using the software and discussing its contents in chatrooms, students were asked to answer an evaluation survey about the whole activity and the usefulness of the software within the learning process. The teaching assistants (TA) evaluated the software as a tool to help in the teaching process. The students' and TAs' satisfaction was very evident and encouraged us to move forward with the software development and to improve the use of this kind of educational tool in biochemistry classes.

Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca(2+)) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca(2+) channels and sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca(2+) channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca(2+) channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca(2+) was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca(2+) channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca(2+) channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca(2+) protein levels. J. Cell. Physiol. 226: 2934-2942, 2011. (C) 2011 Wiley-Liss, Inc.

Cardiac and peripheral adjustments induced by early exercise training intervention were associated with autonomic improvement in infarcted rats: role in functional capacity and mortality

Aims To test the effects of early exercise training (ET) on left ventricular (LV) and autonomic functions, haemodynamics, tissues blood flows (BFs), maximal oxygen consumption (VO(2) max), and mortality after myocardial infarction (MI) in rats. Methods and results Male Wistar rats were divided into: control (C), sedentary-infarcted (SI), and trained-infarcted (TI). One week after MI, TI group underwent an ET protocol (90 days, 50-70% VO2 max). Left ventricular function was evaluated noninvasively and invasively. Baroreflex sensitivity, heart rate variability, and pulse interval were measured. Cardiac output (CO) and regional BFs were determined using coloured microspheres. Infarcted area was reduced in TI (19 +/- 6%) compared with SI (34 +/- 5%) after ET. Exercise training improved the LV and autonomic functions, the CO and regional BF changes induced by MI, as well as increased SERCA2 expression and mRNA vascular endothelial growth factor levels. These changes brought about by ET resulted in mortality rate reduction in the TI (13%) group compared with the SI (54%) group. Conclusion Early aerobic ET reduced cardiac and peripheral dysfunctions and preserved cardiovascular autonomic control after MI in trained rats. Consequently, these ET-induced changes resulted in improved functional capacity and survival after MI.

Angiotensin receptor blockade improves the net balance of cardiac Ca(2+) handling-related proteins in sympathetic hyperactivity-induced heart failure

Aims: The clinical benefits of angiotensin II type 1 (AT1) receptor blockers (ARB) in heart failure (HF) include cardiac anti-remodeling and improved ventricular function. However, the cellular mechanisms underlying the benefits of ARB on ventricular function need to be better clarified. In the present manuscript, we evaluated the effects of AT1 receptor blockade on the net balance of Ca(2+) handling proteins in hearts of mice lacking alpha(2A) and alpha(2C) adrenoceptors (alpha(2A)/alpha(2C)ARKO), which develop sympathetic hyperactivity (SH) induced-HF. Main methods: A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)ARKO mice in a C57BL6/J genetic background (5-7 mo of age) was randomly assigned to receive either placebo or ARB (Losartan, 10 mg/kg for 8wks). Ventricular function (VF) was assessed by echocardiography, and cardiac myocyte width and ventricular fibrosis by a computer-assisted morphometric system. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), phospholamban (PLN), phospho-Ser(16)-PLN, phospho-Thr(17)-PLN, phosphatase 1 (PP1), Na(+)-Ca(2+) exchanger (NCX), Ca(2+)/calmodulin-dependent protein kinase 11 (CaMKII) and phospho-Thr(286)-CaMKII were analyzed by Western blot. Key findings: alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis paralleled by decreased SERCA2 and increased phospho-Thr(17)-PLN, CaMKII, phospho-Thr(286)-CaMKII and NCX levels. ARB induced anti-cardiac remodeling effect and improved VF in alpha(2A)/alpha(2C)ARKO associated with increased SERCA2 and phospho-Ser(16)-PLN levels, and SERCA2:NCX ratio. Additionally, ARB decreased phospho-Thr(17)-PLN levels as well as reestablished NCX, CaMKII and phospho-Thr(286)-CaMKII toward WT levels. Significance: Altogether, these data provide new insights on intracellular Ca(2+) regulatory mechanisms underlying improved ventricular function by ARB therapy in HF. (c) 2011 Elsevier Inc. All rights reserved.

Ischaemic preconditioning improves proteasomal activity and increases the degradation of delta PKC during reperfusion

The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilon PKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished delta PKC translocation by 3.8-fold and increased epsilon PKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of delta PKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilon PKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilon PKC during IPC restored delta PKC levels at the mitochondria while decreasing epsilon PKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a delta PKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, delta PKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilon PKC.

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats

DA SILVA, A. S. R., J. R. PAULI, E. R. ROPELLE, A. G. OLIVEIRA, D. E. CINTRA, C. T. DE SOUZA, L. A. VELLOSO, J. B. C. CARVALHEIRA, and M. J. A. SAAD. Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats. Med. Sci. Sports Exerc., Vol. 42, No. 12, pp. 2180-2188, 2010. Purpose: To evaluate the effects of intensity of exercise on insulin resistance and the expression of inflammatory proteins in the skeletal muscle of diet-induced obese (DIO) rats after a single bout of exercise. Methods: In the first exercise protocol, the rats swam for two 3-h bouts, separated by a 45-min rest period (with 6 h in duration-DIO + EXE), and in the second protocol, the rats were exercised with 45 min of swimming at 70% of the maximal lactate steady state-MLSS (DIO + MLSS). Results: Our data demonstrated that both protocols of exercise increased insulin sensitivity and increased insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate 1 and serine phosphorylation of protein kinase B in the muscle of DIO rats by the same magnitude. In parallel, both exercise protocols also reduced protein tyrosine phosphatase 1B activity and insulin receptor substrate 1 serine phosphorylation, with concomitant reduction in c-jun N-terminal kinase and I kappa B kinase activities in the muscle of DIO rats in a similar fashion. Conclusions: Thus, our data demonstrate that either exercise protocols with low intensity and high volume or exercise with moderate intensity and low volume represents different strategies to restore insulin sensitivity with the same efficacy.

Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like receptor-2 expression as potential targets

Ticks (Acari: Ixodidae) are bloodsucking ectoparasitic arthropods of human and veterinary medical importance. Tick saliva has been shown to contain a wide range of bioactive molecules with vasodilatory, antihemostatic, and immunomodulatory activities. We have previously demonstrated that saliva from Rhipicephalus sanguineus ticks inhibits the maturation of dendritic cells (DCs) stimulated with LPS. Here we examined the mechanism of this immune subversion, evaluating the effect of tick saliva on Toll-like receptor (TLR)-4 signalling pathway in bone marrow-derived DCs. We demonstrated that R. sanguineus tick saliva impairs maturation of DCs stimulated with LIPS, a TLR-4 ligand, leading to increased production of interleukin (IL)-10 and reduced synthesis of IL-12p70 and TNF-alpha. The immunomodulatory effect of the tick saliva on the production of pro-inflammatory cytokines by DCs stimulated with LPS was associated with the observation that tick saliva inhibits the activation of the ERK 1/2 and p38 MAP kinases. These effects were independent of the expression of TLR-4 on the surface of DCs. Additionally, saliva-treated DCs also presented a similar pattern of cytokine modulation in response to other TLR ligands. Since the recent literature reports that several parasites evade immune responses through TLR-2-mediated production of IL-10, we evaluated the effect of tick saliva on the percentage of TLR-2(+) DCs stimulated with the TLR-2 ligand lipoteicoic acid (LTA). The data showed that the population of DCs expressing TLR-2 was significantly increased in DCs treated with LTA plus saliva. In addition, tick saliva alone increased the expression of TLR-2 in a dose- and time-dependent manner. Our data suggest that tick saliva induces regulatory DCs, which secrete IL-10 and low levels of IL-12 and TNF-alpha when stimulated by TLR ligands. Such regulatory DCs are associated with expression of TLR-2 and inhibition of ERK and p38, which promotes the production of IL-10 and thus down-modulates the host`s immune response, possibly favouring susceptibility to tick infestations. (C) 2009 Elsevier B.V. All rights reserved.

Requirement for PLC gamma 2 in IL-3 and GM-CSF-Stimulated MEK/ERK Phosphorylation in Murine and Human Hematopoietic Stem/Progenitor Cells

Even though the involvement of intracellular Ca(2+) (Ca(i)(2+)) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3)) levels and Ca(i)(2+) release in murine and human hematopoietic stem/ progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+)-dependent kinases in MEK activation. In addition, we identify phospholipase C gamma 2 (PLC gamma 2) as a PLC gamma responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCg inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLC gamma 2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/ progenitor cells. J. Cell. Physiol. 226: 1780-1792, 2011. (C) 2010 Wiley-Liss, Inc.

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC(50)). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E(max) = 240 s) is similar to measured for SNP (210 s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1 mu M). In a similar way, 1 mu M ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ. (C) 2011 Elsevier Inc. All rights reserved.

Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The Delta aifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 mu M FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 mu M FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.