Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10 -100 g/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38 MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 g/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) 1 and cardiorespiratory morbidity and mortality (1-3). Even though the relative risks are small, there is considerable public health concern because of the large number of exposed people and the existence of high risk groups. People suffering from asthma constitute a susceptible group, as exemplified by acute symptomatic flares after a sudden surge in ambient PM levels (3). This is likely the result of PM-induced airway inflammation and airway hyperreactivity (4 -10). In addition to these short term effects, animal and human studies conducted with diesel exhaust particles (DEP) as a model air pollutant showed that these particles can enhance allergen-specific IgE production and airway allergic inflammation in parallel with increased Th2 cytokine production (5,(11)(12)(13)(14). This raises the important question of the mechanism of these adverse health effects. Although the biological hypotheses for the mechanisms of PM action are just beginning to develop (15), most of the limited mechanistic data generated to date suggest that oxidative stress is a key biological event in causing the a...

A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However, only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels. However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

The hypoglycemia seen in the fasting PPAR␣ null mouse is thought to be due to impaired liver fatty acid ␤-oxidation. The etiology of hypoglycemia in the PPAR␣ null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPAR␣ null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-13 C 6 ]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-13 C 3 ]lactate or [2-13 C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPAR␣ null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPAR␣ null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-13 C 3 ]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPAR␣ null mice. In contrast, when [2-13 C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPAR␣ null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPAR␣ null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPAR␣ null mice than in controls. In conclusion, PPAR␣ receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.The transition through the fasting and refeeding cycle is an important metabolic adaptation in response to food availability and food intake. The adaptation allows the optimization of fuel energy substrate utilization switching from a glucose-and-fatty acid oxidation to a glucose-and-fatty acid storage state. It is believed that these metabolic changes are effected by the reciprocal action of insulin and glucagon. However, the recent availability of animals with specific nuclear receptor disorders, such as the PPAR␣ KO mice, have drawn attention to the linkage between the regulation of fatty acids by the nuclear receptor family and glucose metabolism. PPAR␣ is a member of the nuclear hormone receptors, the peroxisome-proliferatoractivated receptors (PPARs), 1 for which fatty acids are ligands (reviewed in Refs. 1, 2). PPAR␣ controls the expression of a number of genes involved in mitochondrial and peroxisomal ␤-oxidation and plays an important role in maintaining energy homeostasis (reviewed in Refs. 1, 2) during fasting. PPAR␣ KO mice, fa...

Perineural invasion (PNI) is a common feature of pancreatic ductal adenocarcinoma (PDAC).Here, we investigated the effect of PNI on the microenvironment and how this affects PDAC progression. Transcriptome expression profiles of PDAC tissues with different PNI status were compared, and the intratumoral T cell density and levels of neurotransmitters in these tissues were assessed. PNI was associated with impaired immune responses characterized by decreased CD8+ T and Th1 cells, and increased Th2 cells. Acetylcholine levels were elevated in severe PNI.Acetylcholine impaired the ability of PDAC cells to recruit CD8+ T cells via HDAC1-mediated suppression of CCL5. Moreover, acetylcholine directly inhibited IFN-γ production by CD8+ T cells in a dose-dependent manner, and favored Th2 over Th1 differentiation. Furthermore, hyperactivation of cholinergic signaling enhanced tumor growth by suppressing the intratumoral T cell response in an orthotopic PDAC model. Conversely, blockingResearch.