In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 mu M; 2 h) and subsequently incubated with LPS (5 mu g/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or beta(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor kappa B (NF-kappa B) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

Intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene is associated with decreased NO production in a mercury-exposed population

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a potent vasodilator and plays a prominent role in regulating the cardiovascular system. Decreased basal NO release may predispose to cardiovascular diseases. Evidence suggests that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene may regulate eNOS expression. On the other hand, some recent reports strongly suggest an association between methylmercury (MeHg) exposures and altered NO synthesis. In the present study, we investigate the contribution of the 27-pb tandem repeat polymorphism on nitric oxide production, which could enhance susceptibility to cardiovascular disease in the MeHg-exposed study population. Two-hundred-two participants (98 men and 104 women), all chronically exposed to MeHg through fish consumption were examined. Mean blood Hg concentration and nitrite plasma concentration were 50.5 +/- 35.4 mu g/L and 251.4 +/- 106.3 nM, respectively. Mean systolic and diastolic blood pressure were 120.1 +/- 19.4 mm Hg and 72.0 +/- 10.6 mm Hg, respectively. Mean body mass index was 24.5 +/- 4.3 kg/m(2) and the mean heart rate was 69.8 +/- 11.8 bpm. There were no significant differences in age, arterial blood pressure, body mass index or cardiac frequency between genotype groups (all P>0.05). However, we observed different nitrite concentrations in the genotypes groups, with lower nitrite levels for the 4a4a genotype carriers. Age, gender and the presence of intron 4 polymorphism contributed to nitrite reduction as a result of blood Hg concentration. Taken together, our results show that the 27 nt repeat polymorphism of the intron 4 in the eNOS gene increases susceptibility to cardiovascular diseases after MeHg exposure by modulating nitric oxide levels. (C) 2011 Elsevier B.V. All rights reserved.

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Increased Glyceride-Glycerol Synthesis in Liver and Brown Adipose Tissue of Rat: In-Vivo Contribution of Glycolysis and Glyceroneogenesis

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (similar to 70 %) as well as in BAT and EAT (similar to 80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

Design and synthesis of a new coumarin-based 'turn-on' fluorescent probe selective for Cu+2

The novel coumarin-based 'turn-on' fluorescent probe (E)-3-(2,5-dimethoxybenzylideneamino)-7-hydroxy-2H-chromen-2-one (MGM) was designed, synthesized, and characterized. This compound shows high selectivity for Cu+2, combined with a large fluorescence enhancement upon binding to Cu2+. Benesi-Hildebrand and Job plots demonstrate that the stoichiometry of the Cu+2 complex formed is 2:1. Preliminary studies employing epifluorescence microscopy demonstrated that Cu+2 could be imaged in human neuroblastoma SH-SY5Y cells treated with MGM. (c) 2012 Elsevier Ltd. All rights reserved.

Simcluster: clustering enumeration gene expression data on the simplex space

Abstract Background Transcript enumeration methods such as SAGE, MPSS, and sequencing-by-synthesis EST "digital northern", are important high-throughput techniques for digital gene expression measurement. As other counting or voting processes, these measurements constitute compositional data exhibiting properties particular to the simplex space where the summation of the components is constrained. These properties are not present on regular Euclidean spaces, on which hybridization-based microarray data is often modeled. Therefore, pattern recognition methods commonly used for microarray data analysis may be non-informative for the data generated by transcript enumeration techniques since they ignore certain fundamental properties of this space. Results Here we present a software tool, Simcluster, designed to perform clustering analysis for data on the simplex space. We present Simcluster as a stand-alone command-line C package and as a user-friendly on-line tool. Both versions are available at: http://xerad.systemsbiology.net/simcluster. Conclusion Simcluster is designed in accordance with a well-established mathematical framework for compositional data analysis, which provides principled procedures for dealing with the simplex space, and is thus applicable in a number of contexts, including enumeration-based gene expression data.

In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism

Abstract Background The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p. Results In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain. Conclusions These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

Gene expression and phenotypic traits of Aggregatibacter actinomycetemcomitans in response to environmental changes

Background and Objective: Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. Material and Methods: Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. Results: The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apaH, omp29, cagE and cdtB) and in adhesion to extracellular matrix protein (emaA). Conclusion: Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential.