Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

beta-ADRENOCEPTORS IN THE MEDIAL AMYGDALOID NUCLEUS MODULATE THE TACHYCARDIAC RESPONSE TO RESTRAINT STRESS IN RATS

In the present study, we investigated the involvement of beta-adrenoceptors in the medial amygdaloid nucleus (MeA) in cardiovascular responses evoked in rats submitted to an acute restraint stress. We first pretreated Wistar rats with the nonselective beta-adrenoceptor antagonist propranolol microinjected bilaterally into the MeA (10, 15, and 20 nmol/100 nL) 10 min before exposure to acute restraint. The pretreatment with propranolol did not affect the blood pressure (BP) increase evoked by restraint. However, it increased the tachycardiac response caused by acute restraint when animals were pretreated with a dose of 15 nmol, without a significant effect on the BP response. This result indicates that beta-adrenoceptors in the MeA have an inhibitory influence on restraint-evoked heart rate (HR) changes. Pretreatment with the selective beta(2)-adrenoceptor antagonist ICI 118,551 (10, 15, and 20 nmol/100 nL) significantly increased the restraint-evoked tachycardiac response after doses of 15 and 20 nmol, an effect that was similar to that observed after the pretreatment with propranolol at a dose of 15 nmol, without a significant effect on the BP response. Pretreatment of the MeA with the selective beta(1)-adrenoceptor antagonist CGP 20712 (10, 15, and 20 nmol/100 nL) caused an opposite effect on the HR response, and a significant decrease in the restraint-evoked tachycardia was observed only after the dose of 20 nmol, without a significant effect on the BP response. Because propranolol is an equipotent antagonist of both beta(1) and beta(2)-adrenoceptors, and opposite effects were observed after the treatment with the higher doses of the selective antagonists ICI 118,551 and CGP 20712, the narrow window in the dose-response to propranolol could be explained by a functional antagonism resulting from the simultaneous inhibition of beta(1) and beta(2)-adrenoceptors by the treatment with propranolol. The present results suggest that beta(2)-adrenoceptors have an inhibitory influence on the restraint-evoked tachycardiac response, whereas beta(1)-adrenoceptors have a facilitatory influence on the restraint-evoked tachycardiac response. (c) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

The functional role of ascending nociceptive control in defensive behavior

Ascending nociceptive control is a novel spino-striato-rostral ventral medulla pain modulation pathway that mediates heterosegmental pain-induced analgesia, i.e., noxious stimulus-induced antinociception. In this study, we used the dorsal immobility response in rats as a model of the defensive responses. We demonstrated that the activation of ascending nociceptive control by peripheral noxious stimulation and spinal AMPA and mGluR1 receptor blockade significantly potentiated the duration of the dorsal immobility response in rats via an opioid-dependent mechanism in the nucleus accumbens. These results demonstrated the functional role of ascending nociceptive control in the modulation of defensive responses and spinal glutamatergic receptors in the dorsal immobility response. The immobility response is an antipredator behavior that reflects the underlying state of fear, and ascending nociceptive control may modulate fear. (c) 2012 Elsevier B.V. All rights reserved.

Increased clopidogrel response is associated with ABCC3 expression: A pilot study

Background: The aim of this study was investigate the relationship between ABCB1 and ABCC3 gene expressions in peripheral blood cells (PBC) and the response to clopidogrel in patients with coronary arterial disease (CAD). Methods: Twenty-six male CAD patients (50-70 years) under treatment with clopidogrel (75 mg/day) for at least 5 days were selected. Blood samples were obtained to evaluate platelet reactivity and ABCB1 and ABCC3 mRNA expression. Platelet reactivity was measured in P2Y12 Reaction Units (PRU) using VerifyNow. RNA was extracted from PBC and mRNA levels were measured by qPCR, using GAPD as a reference gene. Results: Platelet response to clopidogrel was categorized in to PRU quartiles. Individuals with PRU values within the first quartile (Q1, <151 units) were considered good responders, while those who had PRU within the fourth quartile (Q4. PRU>260) were considered non-responders. ABCC3 was 1.7 times more expressed in Q4 than in Q1 PRU group (p=0.048). Moreover, CAD patients with low ABCC3 expression (Qe1, <2.5x10(-3)) had higher probability to have a good response to clopidogrel (OR: 18.00, 95%CI: 1.90-169.99, p=0.001). Univariate linear regression analysis demonstrated that low ABCC3 mRNA expression contributed with a reduction of 73 PRU in relation to the patients with expression value higher than 2.5x10(-3) (p=0.027). Neither ABCB1 mRNA levels nor clinical variables studied influenced PRU values. Conclusions: Low ABCC3 mRNA expression in peripheral blood cells is associated with increased clopidogrel response, but further studies are needed to describe the functional relationship of clopidogrel with the ABCC3. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Biventricular structural and functional responses to aortic constriction in a rabbit model of chronic right ventricular pressure overload

Objectives: Chronic right ventricular (RV) pressure overload results in pathologic RV hypertrophy and diminished RV function. Although aortic constriction has been shown to improve systolic function in acute RV failure, its effect on RV responses to chronic pressure overload is unknown. Methods: Adjustable vascular banding devices were placed on the main pulmonary artery and descending aorta. In 5 animals (sham group), neither band was inflated. In 9 animals (PAB group), only the pulmonary arterial band was inflated, with adjustments on a weekly basis to generate systemic or suprasystemic RV pressure at 28 days. In 9 animals, both pulmonary arterial and aortic devices were inflated (PAB+AO group), the pulmonary arterial band as for the PAB group and the aortic band adjusted to increase proximal systolic blood pressure by approximately 20 mm Hg. Effects on the functional performance were assessed 5 weeks after surgery by conductance catheters, followed by histologic and molecular assessment. Results: Contractile performance was significantly improved in the PAB+AO group versus the PAB group for both ventricles. Relative to sham-operated animals, both banding groups showed significant differences in myocardial histologic and molecular responses. Relative to the PAB group, the PAB+AO group showed significantly decreased RV cardiomyocyte diameter, decreased RV collagen content, and reduced RV expression of endothelin receptor type B, matrix metalloproteinase 9, and transforming growth factor beta genes. Conclusions: Aortic constriction in an experimental model of chronic RV pressure overload not only resulted in improved biventricular systolic function but also improved myocardial remodeling. These data suggest that chronically increased left ventricular afterload leads to a more physiologically hypertrophic response in the pressure-overloaded RV. (J Thorac Cardiovasc Surg 2012;144:1494-501)

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Soil-nutrient availability modifies the response of young pioneer and late successional trees to elevated carbon dioxide in a Brazilian tropical environment

The aim of this work was to determine the impact of three levels of [CO2] and two levels of soil-nutrient availability on the growth and physiological responses of two tropical tree species differing in their ecological group: Croton urucurana Baillon, a pioneer (P), and also Cariniana legalis (Martius) Kuntze, a late succession (LS). We aimed to test the hypothesis that P species have stronger response to elevated [CO2] than LS species as a result of differences in photosynthetic capacity and growth kinetics between both functional groups. Seedlings of both species were grown in open-top-chambers under high (HN) or low (LN) soil-nutrient supply and exposed to ambient (380 mu mol mol(-1)) or elevated (570 and 760 mu mol mol(-1)) [CO2]. Measurements of gas exchange, chlorophyll a fluorescence, seedling biomass and allocation were made after 70 days of treatment. Results suggest that elevated [CO2] significantly enhances the photosynthetic rates (A) and biomass production in the seedlings of both species, but that soil-nutrient supply has the potential to modify the response of young tropical trees to elevated [CO2]. In relation to plants grown in ambient [CO2], the P species grown under 760 mu mol mol(-1) [CO2] showed increases of 28% and 91% in A when grown in LN and HN, respectively. In P species grown under 570 mu mol mol(-1) [CO2], A increased by 16% under HN, but there was no effect in LN. In LS species, the enhancement of A by effect of 760 mu mol mol(-1) [CO2] was 30% and 70% in LN and HN, respectively. The exposure to 570 mu mol mol(-1) [CO2] stimulated A by 31% in HN, but was no effect in LN. Reductions in stomatal conductance (g(s)) and transpiration (E), as a result of elevated [CO2] were observed. Increasing the nutrient supply from low to high increased both the maximum rate of carboxylation (V-cmax) and maximum potential rate of electron transport (J(max)). As the level of [CO2] increased, both the V-cmax and the J(max) were found to decrease, whereas the J(max)/V-cmax ratio increased. In the LS species, the maximum efficiency of PSII (F-v/F-m) was higher in the 760 mu mol mol(-1) [CO2] treatment relative to other [CO2] treatments. The results suggest that when grown under HN and the highest [CO2], the performance of the P species C. urucurana, in terms of photosynthesis and biomass enhancement, is better than the LS species C. legalis. However, a larger biomass is allocated to roots when C. legalis seedlings were exposed to elevated [CO2]. This response would be an important strategy for plant survival and productivity of the LS species under drought stresses conditions on tropical environments in a global-change scenario. (C) 2011 Elsevier B.V. All rights reserved.

Modulation of the oscillatory mechanics of lung tissue and the oxidative stress response induced by arginase inhibition in a chronic allergic inflammation model

Abstract Background The importance of the lung parenchyma in the pathophysiology of asthma has previously been demonstrated. Considering that nitric oxide synthases (NOS) and arginases compete for the same substrate, it is worthwhile to elucidate the effects of complex NOS-arginase dysfunction in the pathophysiology of asthma, particularly, related to distal lung tissue. We evaluated the effects of arginase and iNOS inhibition on distal lung mechanics and oxidative stress pathway activation in a model of chronic pulmonary allergic inflammation in guinea pigs. Methods Guinea pigs were exposed to repeated ovalbumin inhalations (twice a week for 4 weeks). The animals received 1400 W (an iNOS-specific inhibitor) for 4 days beginning at the last inhalation. Afterwards, the animals were anesthetized and exsanguinated; then, a slice of the distal lung was evaluated by oscillatory mechanics, and an arginase inhibitor (nor-NOHA) or vehicle was infused in a Krebs solution bath. Tissue resistance (Rt) and elastance (Et) were assessed before and after ovalbumin challenge (0.1%), and lung strips were submitted to histopathological studies. Results Ovalbumin-exposed animals presented an increase in the maximal Rt and Et responses after antigen challenge (p<0.001), in the number of iNOS positive cells (p<0.001) and in the expression of arginase 2, 8-isoprostane and NF-kB (p<0.001) in distal lung tissue. The 1400 W administration reduced all these responses (p<0.001) in alveolar septa. Ovalbumin-exposed animals that received nor-NOHA had a reduction of Rt, Et after antigen challenge, iNOS positive cells and 8-isoprostane and NF-kB (p<0.001) in lung tissue. The activity of arginase 2 was reduced only in the groups treated with nor-NOHA (p <0.05). There was a reduction of 8-isoprostane expression in OVA-NOR-W compared to OVA-NOR (p<0.001). Conclusions In this experimental model, increased arginase content and iNOS-positive cells were associated with the constriction of distal lung parenchyma. This functional alteration may be due to a high expression of 8-isoprostane, which had a procontractile effect. The mechanism involved in this response is likely related to the modulation of NF-kB expression, which contributed to the activation of the arginase and iNOS pathways. The association of both inhibitors potentiated the reduction of 8-isoprostane expression in this animal model.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

Abstract Background Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting. Results High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels. Conclusion The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.

Differential regulation of PGC-1α expression in rat liver and skeletal muscle in response to voluntary running

Abstract Background The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1α. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1α protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism. Methods Two groups of male Wistar rats (2 Mo of age, 188.82 ± 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1α protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations. Results Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean ± SE) of 4.102 ± 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1α protein expression increased significantly from a 1.11 ± 0.12 in the sedentary rats to 1.74 ± 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1α protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1α protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle. Conclusion These data suggest that PGC-1α most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.

Exome analysis of HIV patients submitted to dendritic cells therapeutic vaccine reveals an association of CNOT1 gene with response to the treatment

INTRODUCTION: With the aim of searching genetic factors associated with the response to an immune treatment based on autologous monocyte-derived dendritic cells pulsed with autologous inactivated HIV, we performed exome analysis by screening more than 240,000 putative functional exonic variants in 18 HIV-positive Brazilian patients that underwent the immune treatment. METHODS: Exome analysis has been performed using the ILLUMINA Infinium HumanExome BeadChip. zCall algorithm allowed us to recall rare variants. Quality control and SNP-centred analysis were done with GenABEL R package. An in-house implementation of the Wang method permitted gene-centred analysis. RESULTS: CCR4-NOT transcription complex, subunit 1 (CNOT1) gene (16q21), showed the strongest association with the modification of the response to the therapeutic vaccine (p=0.00075). CNOT1 SNP rs7188697 A/G was significantly associated with DC treatment response. The presence of a G allele indicated poor response to the therapeutic vaccine (p=0.0031; OR=33.00; CI=1.74-624.66), and the SNP behaved in a dominant model (A/A vs. A/G+G/G p=0.0009; OR=107.66; 95% CI=3.85-3013.31), being the A/G genotype present only in weak/transient responders, conferring susceptibility to poor response to the immune treatment. DISCUSSION: CNOT1 is known to be involved in the control of mRNA deadenylation and mRNA decay. Moreover, CNOT1 has been recently described as being involved in the regulation of inflammatory processes mediated by tristetraprolin (TTP). The TTP-CCR4-NOT complex (CNOT1 in the CCR4-NOT complex is the binding site for TTP) has been reported as interfering with HIV replication, through post-transcriptional control. Therefore, we can hypothesize that genetic variation occurring in the CNOT1 gene could impair the TTP-CCR4-NOT complex, thus interfering with HIV replication and/or host immune response. CONCLUSIONS: Being aware that our findings are exclusive to the 18 patients studied with a need for replication, and that the genetic variant of CNOT1 gene, localized at intron 3, has no known functional effect, we propose a novel potential candidate locus for the modulation of the response to the immune treatment, and open a discussion on the necessity to consider the host genome as another potential variant to be evaluated when designing an immune therapy study