Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ)

The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local (125)I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK(1) and NK(2)) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK(1) receptor antagonist, and in a lesser extent by SR48968, an NK(2) antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK(1) and NK(2) receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

Critical involvement of Th1-related cytokines in renal injuries induced by ischemia and reperfusion

Renal ischemia and reperfusion injury (IRI) is considered an inflammatory syndrome. To move forward in its pathogenesis, we exploited the role of several cytokines on renal damages triggered by IRI. Specifically to evaluate the role of Th1 immune profile in this system, IL-12, IFN-gamma, and IFN-gamma/IL-12 deficient (KO) mice on C57BL/6 background and their controls were subjected to IRI. In each group, blood and kidney samples were harvested. Renal function was evaluated by serum creatinine and renal morphometric analyses. Gene expression of IL-6 and HO-1 were also investigated by Q-PCR. IFN-gamma KO animals presented the highest impairment in renal function compared to controls. Conversely, IL-12 KO animals were absolutely protected and, in a lesser extent, IFN-gamma/IL-12 KO double knockout was also protected from IRI. Gene expression analyses showed higher expression of HO-1, a cytoprotective gene, and IL-6, a pro-inflammatory cytokine, in IFN-gamma deficient animals subjected to IRI. Our results confirm that Th1 related cytokines such as IL-12 and IFN-gamma are critically involved in renal ischemia and reperfusion injury. (C) 2008 Elsevier B.V. All rights reserved.

Osteopontin involvement in granuloma formation and in the severity of Paracoccidioides brasiliensis infection

The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN(+) cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Co-stressors chilling and high light increase photooxidative stress in diuron-treated red alga Kappaphycus alvarezii but with lower involvement of H(2)O(2)

Diuron is one of the most commonly found N-phenylurea herbicides in marine/estuarine waters that promotes toxic effects by inhibiting photosynthesis and affecting the production of reactive oxygen species (ROS) in autotrophs. Since photo- and thermoacclimation are also ROS-mediated processes, this work evaluates a hypothetical additive effect of high light (HL) and chilling (12 degrees C) on 50 nM diuron toxicity to the highly-photosynthetically active apices of the red alga Kappaphycus alvarezii. Additive inhibition of photosynthesis was mainly evidenced by significant decreases of quantum yield of photosystem II and electron transfer rates upon co-stressors exposure to diuron-treated algae. Under extreme 12 degrees C/HL/diuron conditions, unexpected lower correlations between H(2)O(2) concentrations in seawater and radical-sensitive protein thiols were concomitantly measured with the highest indexes of photoinhibition (parameter beta). Altogether, these data support the hypothesis that co-stressors chilling/HL additively inhibit photosynthesis in diuron-exposed K. alvarezii but with less involvement of H(2)O(2) in injury effects than with only chilling or HL. (C) 2010 Elsevier Inc. All rights reserved.

Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia

Schizophrenia is likely to be a consequence of serial alterations in a number of genes that, together with environmental factors, will lead to the establishment of the illness. The dorsolateral prefrontal cortex (Brodmann`s Area 46) is implicated in schizophrenia and executes high functions such as working memory, differentiation of conflicting thoughts, determination of right and wrong concepts, correct social behavior and personality expression. We performed a comparative proteome analysis using two-dimensional gel electrophoresis of pools from 9 schizophrenia and 7 healthy control patients` dorsolateral prefrontal cortex aiming to identify, by mass spectrometry, alterations in protein expression that could be related to the disease. In schizophrenia-derived samples, our analysis revealed 10 downregulated and 14 upregulated proteins. These included alterations previously implicated in schizophrenia, such as oligodendrocyte-related proteins (myelin basic protein and transferrin), as well as malate dehydrogenase, aconitase, ATP synthase subunits and cytoskeleton-related proteins. Also, six new putative disease markers were identified, including energy metabolism, cytoskeleton and cell signaling proteins. Our data not only reinforces the involvement of proteins previously implicated in schizophrenia, but also suggests new markers, providing further information to foster the comprehension of this important disease. (C) 2008 Elsevier Ltd. All rights reserved.

Involvement of free radicals in peroxidatic reactions catalyzed by chloroperoxidase

The mechanism of chloroperoxidase (CPO)-catalyzed peroxidatic reactions of several substituted hydroquinones was studied at various hydrogen peroxide concentrations. The pathway was studied using cytochrome c as the radical trapping agent. As the hydroquinones became more hindered there was a difference in the amount of radicals trapped. For hydroquinone, 59.3% radical pathway, and methylhydroquinone, 81.4% radical, the difference in radicals trapped is due to a difference in pathway. For 2,3-dimethylhydroquinone (75.4%), trimethylhydroquinone (44.5%), and t-butylhydroquinone (0%) other non-peroxidatic reactions are noticed. Thus, for the more substituted hydroquinones the difference in radicals trapped can not be assigned to a difference in radical pathway. Also, there were problems drawing conclusions for this system due to the catalytic reaction of hydrogen peroxide. The radical trapping ability of 2,4,6-trimethylphenol was investigated for various other substrates. TMP reacted with the radicals generated in the enzymatic reactions of phenol, resorcinol, and m-methoxyphenol. Thus, this TMP system offers further potential as another radical trapping agent for use in these studies.