Ethanol stress decreases the expression of IBP in bacteria.

Fine-tuning in regulation of Clp protein content in Bacillus subtilis.

Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis.

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen.

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes

Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis. We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate- but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipid-induced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Leptin deficiency contributes to the pathogenesis of alcoholic fatty liver disease in mice.

White adipose tissue (WAT) secretes adipokines, which critically regulate lipid metabolism. The present study investigated the effects of alcohol on adipokines and the mechanistic link between adipokine dysregulation and alcoholic fatty liver disease. Mice were fed alcohol for 2, 4, or 8 weeks to document changes in adipokines over time. Alcohol exposure reduced WAT mass and body weight in association with hepatic lipid accumulation. The plasma adiponectin concentration was increased at 2 weeks, but declined to normal at 4 and 8 weeks. Alcohol exposure suppressed leptin gene expression in WAT and reduced the plasma leptin concentration at all times measured. There is a highly positive correlation between plasma leptin concentration and WAT mass or body weight. To determine whether leptin deficiency mediates alcohol-induced hepatic lipid dyshomeostasis, mice were fed alcohol for 8 weeks with or without leptin administration for the last 2 weeks. Leptin administration normalized the plasma leptin concentration and reversed alcoholic fatty liver. Alcohol-perturbed genes involved in fatty acid β-oxidation, very low-density lipoprotein secretion, and transcriptional regulation were attenuated by leptin. Leptin also normalized alcohol-reduced phosphorylation levels of signal transducer Stat3 and adenosine monophosphate-activated protein kinase. These data demonstrated for the first time that leptin deficiency in association with WAT mass reduction contributes to the pathogenesis of alcoholic fatty liver disease.

Diminished sympathetic nervous system activity in genetically obese (ob/ob) mouse.

The genetically obese (ob/ob) mouse exhibits defective thermoregulatory responses to cold exposure. Pathophysiological explanations for this phenomenon have focused on abnormalities in intracellular metabolism or insensitivity of peripheral tissues to the thermogenic effects of catecholamines. Because the sympathetic nervous system (SNS) is subject to feedback regulation, a peripheral impairment in thermogenesis should be associated with a compensatory increase in SNS activity. To examine SNS activity in the ob/ob mouse, norepinephrine (NE) turnover was measured in heart and interscapular brown adipose tissue (IBAT) of ob/ob and lean mice. The results from studies utilizing radiolabeled NE or inhibition of NE biosynthesis with alpha-methyl-p-tyrosine to measure NE turnover demonstrated reductions in SNS activity of 33-56% in heart and of 45-73% in IBAT in ob/ob mice at ambient temperature (22 degrees C) compared with measurements in lean controls. During cold exposure (4 degrees C) NE turnover increased in heart and IBAT to a similar extent in both ob/ob and lean mice, but NE turnover rates in heart, and probably in IBAT as well, remained lower in the obese mice than in the lean despite the gradual development of hypothermia in the ob/ob mice during this period. Administration of naltrexone, a long-acting opiate antagonist, failed to reverse the suppression of SNS activity observed in the ob/ob mice. These data indicate that diminished SNS activity in ob/ob mice may be an additional factor contributing to the defective thermogenesis characteristic of these animals.

Acetic Acid Activates the AMP-Activated Protein Kinase Signaling Pathway to Regulate Lipid Metabolism in Bovine Hepatocytes

The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPKα inhibitor). Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 107 plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-β (IFN-β) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2’, 5’-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1β (IL-1β) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-β and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1.

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-α/β and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3′ non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5′ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP–RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.

Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking.

Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the alpha4beta2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the alpha4beta2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli

Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

OBJECTIVE Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. STUDY DESIGN Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. RESULTS Corin concentrations were significantly higher in mild (2.78 ± 0.67 ng/ml, p < .05) and severe (2.53 ± 0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ± 0.08 ng/ml) and CHT (1.55 ± 0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ± 0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ± 0.24 ng/ml, p < .01) than in normotensive (0.48 ± 0.06 ng/ml) and mild preeclampsia (0.52 ± 0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. CONCLUSIONS ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.

Crucial Role of Interferon Consensus Sequence Binding Protein, but neither of Interferon Regulatory Factor 1 nor of Nitric Oxide Synthesis for Protection Against Murine Listeriosis

Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF)1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP−/− and to a lesser degree also IRF2−/− mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-γ in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-γ stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-γ–mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response.

The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1Δ strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR.

Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury.

Kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), are associated with inflammation. The mechanism that regulates inflammation in these renal injuries remains unclear. Here, we demonstrated that p300/CBP-associated factor (PCAF), a histone acetyltransferase, was overexpressed in the kidneys of db/db mice and lipopolysaccharide (LPS)-injected mice. Moreover, elevated histone acetylation, such as H3K18ac, and up-regulation of some inflammatory genes, such as ICAM-1, VCAM-1, and MCP-1, were found upon these renal injuries. Furthermore, increased H3K18ac was recruited to the promoters of ICAM-1, VCAM-1, and MCP-1 in the kidneys of LPS-injected mice. In vitro studies demonstrated that PCAF knockdown in human renal proximal tubule epithelial cells (HK-2) led to downregulation of inflammatory molecules, including VCAM-1, ICAM-1, p50 subunit of NF-κB (p50), and MCP-1 mRNA and protein levels, together with significantly decreased H3K18ac level. Consistent with these, overexpression of PCAF enhanced the expression of inflammatory molecules. Furthermore, PCAF deficiency reduced palmitate-induced recruitment of H3K18ac on the promoters of ICAM-1 and MCP-1, as well as inhibited palmitate-induced upregulation of these inflammatory molecules. In summary, the present work demonstrates that PCAF plays an essential role in the regulation of inflammatory molecules through H3K18ac, which provides a potential therapeutic target for inflammation-related renal diseases.

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression.

Chronic variable stress activates hematopoietic stem cells

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk between the brain and immune system includes the hypothalamic-pituitary-adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic-adrenal-medullary axis, which controls stress-induced catecholamine release in support of the fight-or-flight reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic progenitors, giving rise to higher levels of disease-promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae.

In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms.

Nonreplicating and metabolically quiescent bacteria are implicated in latent tuberculosis infections and relapses following "sterilizing" chemotherapy. However, evidence linking bacterial dormancy and persistence in vivo is largely inconclusive. Here we measure the single-cell dynamics of Mycobacterium tuberculosis replication and ribosomal activity using quantitative time-lapse microscopy and a reporter of ribosomal RNA gene expression. Single-cell dynamics exhibit heterogeneity under standard growth conditions, which is amplified by stressful conditions such as nutrient limitation, stationary phase, intracellular replication, and growth in mouse lungs. Additionally, the lungs of chronically infected mice harbor a subpopulation of nongrowing but metabolically active bacteria, which are absent in mice lacking interferon-γ, a cytokine essential for antituberculosis immunity. These cryptic bacterial forms are prominent in mice treated with the antituberculosis drug isoniazid, suggesting a role in postchemotherapeutic relapses. Thus, amplification of bacterial phenotypic heterogeneity in response to host immunity and drug pressure may contribute to tuberculosis persistence.

The RNA Polymerase “Switch Region” Is a Target for Inhibitors

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

Enhancement of anxiety-like responsiveness to the cannabinoid CB(1) receptor agonist HU-210 following chronic stress.

The effect that chronic unpredictable stress had on the anxiety-like response elicited by the cannabinoid receptor agonist HU-210 [3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol] in the elevated plus maze was investigated here. Male Long-Evans rats were either unstressed or were subjected to a 21-day regimen of chronic unpredictable stress, and subsequently were subdivided into three testing groups (vehicle, 10 and 50 microg/kg of HU-210) and tested on the elevated plus maze. Results demonstrated that in unstressed animals, a low dose of HU-210 induced an anxiolytic response, whereas a high dose induced an anxiogenic response. Further, in stressed animals both the low and the high doses of HU-210 induced anxiogenic responses. These findings suggest that chronic stress enhances either cannabinoid receptor responsivity or one of the interacting systems implicated in emotional states.

Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p.

Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

Effect of cyclic AMP-dependent protein phosphorylating conditions on the pH-dependent activity of tyrosine hydroxylase from beef and rat striata.

Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23-fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP[-dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G-25 Sephadex-treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP-dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Exploiting the yeast stress-activated signaling network to inform on stress biology and disease signaling

Healthy cells utilize intricate systems to monitor their environment and mount robust responses in the event of cellular stress. Whether stress arises from external insults or defects due to mutation and disease, cells must be able to respond precisely to mount the appropriate defenses. Multi-faceted stress responses are generally coupled with arrest of growth and cell-cycle progression, which both limits the transmission of damaged materials and serves to reallocate limited cellular resources toward defense. Therefore, stress defense versus rapid growth represent competing interests in the cell. How eukaryotic cells set the balance between defense versus proliferation, and in particular knowledge of the regulatory networks that control this decision, are poorly understood. In this perspective, we expand upon our recent work inferring the stress-activated signaling network in budding yeast, which captures pathways controlling stress defense and regulators of growth and cell-cycle progression. We highlight similarities between the yeast and mammalian stress responses and explore how stress-activated signaling networks in yeast can inform on signaling defects in human cancers.

Rejuvenation of aged progenitor cells by exposure to a young systemic environment

The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-α and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-α complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.

Naringenin inhibits allergen-induced airway inflammation and airway responsiveness and inhibits NF-kappaB activity in a murine model of asthma.

Naringenin, a flavonoid, has antiinflammatory and immunomodulatory properties. We investigated whether naringenin could attenuate allergen-induced airway inflammation and its possible mechanism in a murine model of asthma. Mice were sensitized and challenged with ovalbumin. Some mice were administered with naringenin before ovalbumin challenge. We evaluated the development of airway inflammation and airway reactivity. Interleukin (IL)4, IL13, chemokine (C-C motif) ligand (CCL)5, and CCL11 in bronchoalveolar lavage fluid and serum total IgE were detected by ELISA. IkappaBalpha degradation and inducible nitric oxide synthase (iNOS) in lungs were measured by Western blot. We also tested NF-kappaB binding activity by electrophoretic mobility shift assay. The mRNA levels of iNOS, CCL5, and CCL11 were detected by real-time PCR. Naringenin attenuated ovalbumin-induced airway inflammation and airway reactivity in experimental mice. The naringenin-treated mice had lower levels of IL4 and IL13 in the bronchoalveolar lavage fluid and lower serum total IgE. Furthermore, naringenin inhibited pulmonary IkappaBalpha degradation and NF-kappaB DNA-binding activity. The levels of CCL5, CCL11, and iNOS were also significantly reduced. The results indicated that naringenin may play protective roles in the asthma process. The inhibition of NF-kappaB and the decreased expression of its target genes may account for this phenomenon.

Extensive diversity of Ig-superfamily proteins in the immune system of insects.

The extensive somatic diversification of immune receptors is a hallmark of higher vertebrates. However, whether molecular diversity contributes to immune protection in invertebrates is unknown. We present evidence that Drosophila immune-competent cells have the potential to express more than 18,000 isoforms of the immunoglobulin (Ig)-superfamily receptor Down syndrome cell adhesion molecule (Dscam). Secreted protein isoforms of Dscam were detected in the hemolymph, and hemocyte-specific loss of Dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial binding. Importantly, the molecular diversity of Dscam transcripts generated through a mechanism of alternative splicing is highly conserved across major insect orders, suggesting an unsuspected molecular complexity of the innate immune system of insects.

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.

Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response.

Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ33630) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.

Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone.

Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast

A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. This apparently simple auxotroph did not grow well in rich media containing excess methionine, forming small colonies on yeast extract/peptone/dextrose plates. Faster-growing large colonies were abundant when overnight cultures were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide single-nucleotide polymorphism and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Met4 activates and Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three Opi1p-regulated reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.

Macrophages induce COX-2 expression in breast cancer cells: role of IL-1β autoamplification.

Tumor-associated macrophages and high levels of cyclooxygenase-2 (COX-2) are associated with poor prognosis in breast cancer patients, but their potential interdependence has not been evaluated. The objective of this study was to determine whether macrophages regulate COX-2 expression in breast cancer cells. For this purpose, THP-1 cells were cocultured with HCC1954 breast cancer cells. Coculture led to increased COX-2 expression in the HCC1954 cells and elevated prostaglandin E(2) levels in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 breast cancer cells or when human monocyte-derived macrophages were cocultured with HCC1954 cells. Coculture triggered production of reactive oxygen species (ROS) in HCC1954 cells. COX-2 induction was blocked in cells preincubated with an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or by silencing p67PHOX, a subunit of NADPH oxidase. ROS production triggered activation of Src and mitogen-activated protein kinases (MAPKs). Blocking Src or MAPK activities or antagonizing the activator protein-1 (AP-1) transcription factor attenuated COX-2 induction in HCC1954 cells. Coculture caused rapid induction of interleukin-1β (IL-1β) in both breast cancer cells and macrophages. Increased IL-1β expression was blocked by an interleukin-1 receptor antagonist (IL-1Ra), suggesting autocrine and paracrine effects. Importantly, macrophage-induced COX-2 expression was blocked in HCC1954 cells preincubated with IL-1Ra or anti-IL-1β IgG. Together, these results indicate that macrophage-mediated induction of COX-2 in breast cancer cells is a consequence of IL-1β-mediated stimulation of ROS→Src→MAPK→AP-1 signaling. IL-1β-dependent induction of COX-2 in breast cancer cells provides a mechanism whereby macrophages contribute to tumor progression and potential therapeutic targets in breast cancer.

IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice.

Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

Induction of the heat-shock response by carbon dioxide in Chironomus thummi.

The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

Article

IBD is characterized by uncontrolled immune responses in inflamed mucosa, with dominance of IL-17-producing cells and deficiency of Treg cells. The aim of this study was to explore the effect and mechanisms of RA, the ligand of RARalpha, on immune responses in human and murine colitis. Colonic biopsies from patients with UC were cultured and treated with RA as the agonist of RARalpha or LE135 as the antagonist of RARalpha. Expressions of IL-17 and FOXP3 were detected by immunohistochemistry. Murine colitis was induced by intrarectal administration with TNBS at Day 1. Mice were then i.p.-treated with RA or LE135 daily for 7 days. Cytokine levels in the cultures of mouse LPMCs were measured. Expressions of FOXP3 and IL-17 in colon tissues or MLN were detected by immunohistological analysis. Body weight and colon inflammation were evaluated. RA treatment up-regulated FOXP3 expression and down-regulated IL-17 expression in colon biopsies of patients and in colon tissues and MLN of mice with colitis compared with controls. LPMCs from RA-treated mice produced lower levels of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-17) but more regulatory cytokines (IL-10, TGF-beta) compared with that of untreated mice. LE135 showed the opposite effect of RA. Furthermore, RA ameliorated TNBS-induced colitis in a dose-dependent manner, as seen by improved body weight and colon inflammation. RA down-regulates colon inflammatory responses in patients with IBD in vitro and in murine colitis in vivo, representing a potential therapeutic approach in IBD treatment.

Soluble MD-2 is an acute-phase protein and an opsonin for Gram-negative bacteria.

Myeloid differentiation factor-2 (MD-2) is a lipopolysaccharide (LPS)-binding protein usually coexpressed with and binding to Toll-like receptor 4 (TLR4), conferring LPS responsiveness of immune cells. MD-2 is also found as a soluble protein. Soluble MD-2 (sMD-2) levels are markedly elevated in plasma from patients with severe infections, and in other fluids from inflamed tissues. We show that sMD-2 is a type II acute-phase protein. Soluble MD-2 mRNA and protein levels are up-regulated in mouse liver after the induction of an acute-phase response. It is secreted by human hepatocytic cells and up-regulated by interleukin-6. Soluble MD-2 binds to Gram-negative but not Gram-positive bacteria, and sMD-2 secreted by hepatocytic cells is an essential cofactor for the activation of TLR4-expressing cells by Gram-negative bacteria. Soluble MD-2 opsonization of Gram-negative bacteria accelerates and enhances phagocytosis, principally by polymorphonuclear neutrophils. In summary, our results demonstrate that sMD-2 is a newly recognized type II acute-phase reactant, an opsonin for Gram-negative bacteria, and a cofactor essential for the activation of TLR4-expressing cells. This suggests that sMD-2 plays a key role in the host innate immune response to Gram-negative infections.

Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size

How the number of immune cells recruited to sites of infection is determined and adjusted to differences in the cellular stoichiometry between host and pathogen is unknown. Here, we have uncovered a role for reactive oxygen species (ROS) as sensors of microbe size. By sensing the differential localization of ROS generated in response to microbes of different size, neutrophils tuned their interleukin (IL)-1β expression via the selective oxidation of NF-κB, in order to implement distinct inflammatory programs. Small microbes triggered ROS intracellularly, suppressing IL-1β expression to limit neutrophil recruitment as each phagocyte eliminated numerous pathogens. In contrast, large microbes triggered ROS extracellularly, amplifying IL-1β expression to recruit numerous neutrophils forming cooperative clusters. Defects in ROS-mediated microbe size sensing resulted in large neutrophil infiltrates and clusters in response to small microbes that contribute to inflammatory disease. These findings highlight the impact of ROS localization on signal transduction.

Up-regulation of endothelial nitric-oxide synthase by endothelium-derived hyperpolarizing factor involves mitogen-activated protein kinase and protein kinase C signaling pathways.

Cytochrome P450 (P450)-dependent metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are proposed to be endothelium-derived hyperpolarizing factors (EDHF) that affect vascular tone; however, the effects of EDHF on endothelial-derived nitric oxide biosynthesis remain unknown. We examined the regulation of endothelial nitric-oxide synthase (eNOS) by EDHF and investigated the relevant signaling pathways involved. The P450 epoxygenases CYP102 F87V mutant, CYP2C11-CYPOR, and CYP2J2 were transfected into cultured bovine aortic endothelial cells, and the effects of endogenously formed or exogenously applied EETs on eNOS expression and activity were assessed. Transfection with the P450 epoxygenases led to increased eNOS protein expression, an effect that was attenuated by cotreatment with the P450 inhibitor 17-ODYA. Northern analysis demonstrated that P450 transfection led to increased eNOS mRNA levels consistent with an effect at the pretranslational level. P450 epoxygenase transfection resulted in increased eNOS activity as measured by the conversion of L-arginine to L-citrulline. Addition of synthetic EETs (50-200 nM) to the culture media also increased eNOS expression and activity. Treatment with mitogen-activated protein kinase (MAPK), MAPK kinase, and protein kinase C inhibitors apigenin, 2'-amino-3'-methoxyflavone (PD98059), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), respectively, significantly inhibited the effects of P450 transfection on eNOS expression. Overexpression of P450 epoxygenases or addition of synthetic EETs increased Thr495 phosphorylation of eNOS, an effect that was inhibited by both apigenin and PD98059. Overexpression of P450 epoxygenases in rats resulted in increased aortic eNOS expression, providing direct evidence that EDHF can influence vascular eNOS levels in vivo. Based on this data, we conclude that EDHF up-regulates eNOS via activation of MAPK and protein kinase C signaling pathways.

Expression of optineurin, a glaucoma-linked gene, is influenced by elevated intraocular pressure.

Optineurin (optic neuropathy inducing protein; OPTN) was recently linked to 16.7% of families with primary open-angle glaucoma. The function of OPTN in the eye is not known, but is present in the trabecular meshwork, which is responsible for maintenance of intraocular pressure (IOP). To gain insight into the role of OPTN in the development of glaucoma we studied its expression in response to factors known to be associated with the disease: elevated IOP, tumor necrosis factor-alpha (TNFalpha), and dexamethasone (DEX). We performed the treatments in human organ cultures under conditions mimicking physiological pressure. We find OPTN significantly upregulated after 2, 4, and 7 days of sustained elevated IOP. OPTN expression is also induced 2.3-fold by TNFalpha and 2.6-fold by prolonged DEX treatment. These results demonstrate that OPTN is part of the transcriptome responding to glaucomatous insults and support the protective role of this protein in the trabecular meshwork.

Antioxidant Defenses in the Rat Placenta in Late Gestation: Increased Labyrinthine Expression of Superoxide Dismutases, Glutathione Peroxidase 3, and Uncoupling Protein 21

Placental oxidative stress plays a key role in the pathophysiology of placenta-related disorders, most notably preeclampsia (PE) and intrauterine growth restriction (IUGR). Oxidative stress occurs when accumulation of reactive oxygen species (ROS) damages DNA, proteins and lipids, an outcome that is limited by antioxidant enzymes; mitochondrial uncoupling protein 2 (UCP2) may also limit oxidative stress by reducing ROS production. Here we characterized placental antioxidant defenses during normal gestation and following glucocorticoid-induced IUGR. Placentas were collected on Days 16 and 22 of normal rat pregnancy (term = Day 23) and at Day 22 after dexamethasone treatment from Day 13. Expression of several genes encoding antioxidant enzymes (Sod1, Sod2, Sod3, Cat, Gpx3, Txn1, Txnrd1, Txnrd2, and Txnrd3) and Ucp2 was measured by quantitative RT-PCR in the labyrinth (LZ) and junctional zones (JZ) of the placenta. Expression of Sod1 and Ucp2 mRNAs and the activity of xanthine oxidase, a source of ROS, all increased from Days 16 to 22 in both placental zones, whereas Sod2 and Gpx3 increased only in the rapidly growing LZ. In contrast, Sod3 and Txnrd1 expression fell in the LZ over this period, whereas total superoxide dismutase activity remained stable. Dexamethasone treatment reduced fetal-placental growth and LZ expression of Ucp2 but increased JZ expression of Txn1. Indices of placental oxidative damage (TBARS, F2-isoprostanes, and 8-OHdG) did not change with gestational age or dexamethasone, indicative of adequate antioxidant protection. Overall, our data suggest that the rat placenta is protected from oxidative stress by the dynamic zone- and stage-dependent expression of antioxidant defense genes.

IAP regulation of metastasis.

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.

The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.

The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

Peroxisome proliferator-activated receptor alpha activation decreases metastatic potential of melanoma cells in vitro via down-regulation of Akt.

PURPOSE Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well. We previously reported the antimetastatic activity of the PPARalpha ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action. EXPERIMENTAL DESIGN Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARalpha expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system. RESULTS Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar. These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal-regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model. CONCLUSIONS Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

BACKGROUND & AIMS GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Evaluation of oxidative status and depression-like responses in Brown Norway rats with acute myeloid leukemia

It has been proved that oxidative stress increases when leukemia is accompanied by depression. This fact may indicate the role of oxidative stress in the development of depression in cancer patients. The aim of this study was to determine whether the acute myeloid leukemia of Brown Norway rats, which is accompanied by oxidative stress, evoked behavioral and receptor changes resembling alterations characteristic of rat models of depression. The rats were divided into two groups: leukemic rats and healthy control. Leukemia was induced through intraperitoneal injection of 10(7) promyelocytic leukemia cells to the Brown Norway rats. Depression-like behavior was evaluated in the forced swim test at 30 or 34 days after leukemic cells injection. The rats were killed after the evaluation and the spleen, brain cortex and hippocampus were excised. The red-ox state was assessed in homogenates of tissues by measuring total glutathione (GSH) content, the ferric ion reducing ability of plasma (FRAP) level, expression of heme oxygenase-1 (HO-1), biliverdin reductase (BvR) and ferritin mRNA, superoxide dismutase (SOD) activity, as well as malondialdehyde (MDA) concentration. Radioligand binding assay was used to assess of the effect of leukemia on cortical receptors. Leukemic cells were identified using RM-124 antibody by FACS Calibur flow cytometry. Leukemia influenced locomotory activity as well as forced swim test behavior in a 34-day series of experiments. Signs of oxidative stress in leukemic rats were observed in each examined stage of leukemia development. The FRAP values and glutathione contents, were significantly lowered whereas HO-1 mRNA expression, and malonodialdehyde concentrations were significantly increased in the spleen and brain structures of leukemic rats in comparison with the healthy controls. A significant increase in the potency of glycine to displace [(3)H]L-689,560 from the strychnine-insensitive glycine site of the N-methyl-D-aspartic (NMDA) receptors receptor complex in cortical homogenates of the leukemic rats in 30- and 34-day experimental series was observed in comparison with the control. Upregulation of 5-HT(2A) receptors was observed in rat cortex after 30 days of leukemia development but not in 34-days series compared with the control. It is concluded that disturbances in antioxidant system in brain cortex were accompanied by an activation of glycine sites of the NMDA receptor complex, regardless of stage of leukemia development, which are characteristic of model of depression. Findings of our study demonstrate the link between glutamatergic activity, oxidative stress and leukemia.

Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

Cell death from antibiotics without the involvement of reactive oxygen species.

Recent observations have suggested that classic antibiotics kill bacteria by stimulating the formation of reactive oxygen species (ROS). If true, this notion might guide new strategies to improve antibiotic efficacy. In this study, the model was directly tested. Contrary to the hypothesis, antibiotic treatment did not accelerate the formation of hydrogen peroxide in Escherichia coli and did not elevate intracellular free iron, an essential reactant for the production of lethal damage. Lethality persisted in the absence of oxygen, and DNA repair mutants were not hypersensitive, undermining the idea that toxicity arose from oxidative DNA lesions. We conclude that these antibiotic exposures did not produce ROS and that lethality more likely resulted from the direct inhibition of cell-wall assembly, protein synthesis, and DNA replication.

Identification and Characterization of Multiple TRIM Proteins That Inhibit Hepatitis B Virus Transcription

Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.

Multiple RNA surveillance pathways limit aberrant expression of iron uptake mRNAs and prevent iron toxicity in S. cerevisiae.

Tight regulation of the expression of mRNAs encoding iron uptake proteins is essential to control iron homeostasis and avoid intracellular iron toxicity. We show that many mRNAs encoding iron uptake or iron mobilization proteins are expressed in iron-replete conditions in the absence of the S. cerevisiae RNase III ortholog Rnt1p or of the nuclear exosome component Rrp6p. Extended forms of these mRNAs accumulate in the absence of Rnt1p or of the 5'-->3' exonucleases Xrn1p and Rat1p, showing that multiple degradative pathways contribute to the surveillance of aberrant forms of these transcripts. RNase III-deficient cells are hypersensitive to high iron concentrations, suggesting that Rnt1p-mediated RNA surveillance is required to prevent iron toxicity. These results show that RNA surveillance through multiple ribonucleolytic pathways plays a role in iron homeostasis in yeast to avoid the potentially toxic effects of the expression of the iron starvation response in iron-replete conditions.

Differential role of CbpA and PspA in modulation of in vitro CXC chemokine responses of respiratory epithelial cells to infection with Streptococcus pneumoniae.

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.

Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.

The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.

Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in the Western world. Twenty per cent of NAFLD individuals develop chronic hepatic inflammation (non-alcoholic steatohepatitis, NASH) associated with cirrhosis, portal hypertension and hepatocellular carcinoma, yet the causes of progression from NAFLD to NASH remain obscure. Here, we show that the NLRP6 and NLRP3 inflammasomes and the effector protein IL-18 negatively regulate NAFLD/NASH progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different mouse models reveal that inflammasome-deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, leading to enhanced hepatic tumour-necrosis factor (TNF)-α expression that drives NASH progression. Furthermore, co-housing of inflammasome-deficient mice with wild-type mice results in exacerbation of hepatic steatosis and obesity. Thus, altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of heretofore seemingly unrelated systemic auto-inflammatory and metabolic disorders.

IL-23–responsive innate lymphoid cells are increased in inflammatory bowel disease

Results of experimental and genetic studies have highlighted the role of the IL-23/IL-17 axis in the pathogenesis of inflammatory bowel disease (IBD). IL-23-driven inflammation has been primarily linked to Th17 cells; however, we have recently identified a novel population of innate lymphoid cells (ILCs) in mice that produces IL-17, IL-22, and IFN-γ in response to IL-23 and mediates innate colitis. The relevance of ILC populations in human health and disease is currently poorly understood. In this study, we have analyzed the role of IL-23-responsive ILCs in the human intestine in control and IBD patients. Our results show increased expression of the Th17-associated cytokine genes IL17A and IL17F among intestinal CD3⁻ cells in IBD. IL17A and IL17F expression is restricted to CD56⁻ ILCs, whereas IL-23 induces IL22 and IL26 in the CD56⁺ ILC compartment. Furthermore, we observed a significant and selective increase in CD127⁺CD56⁻ ILCs in the inflamed intestine in Crohn's disease (CD) patients but not in ulcerative colitis patients. These results indicate that IL-23-responsive ILCs are present in the human intestine and that intestinal inflammation in CD is associated with the selective accumulation of a phenotypically distinct ILC population characterized by inflammatory cytokine expression. ILCs may contribute to intestinal inflammation through cytokine production, lymphocyte recruitment, and organization of the inflammatory tissue and may represent a novel tissue-specific target for subtypes of IBD.

Disruption of the 5S RNP–Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2C305F knock-in mice that have a disrupted 5S RNP–Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2C305F reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP–Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP–Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

C-reactive protein decreases interleukin-10 secretion in activated human monocyte-derived macrophages via inhibition of cyclic AMP production.

OBJECTIVE C-Reactive protein (CRP), a cardiovascular risk marker, could also participate in atherosclerosis. Atherosclerotic plaques express CRP and interleukin (IL)-10, a major antiinflammatory cytokine. IL-10 deficiency results in increased lesion formation, whereas IL-10 delivery attenuates lesions. We tested the effect of CRP on lipopolysaccharide (LPS)-induced IL-10 secretion in human monocyte-derived macrophages (HMDMs). METHODS AND RESULTS Incubation of HMDMs with CRP significantly decreased LPS-induced IL-10 mRNA and intracellular and secreted IL-10 protein and destabilized IL-10 mRNA. Also, CRP alone increased secretion of IL-8, IL-6, and tumor necrosis factor from HMDMs and did not inhibit LPS-induced secretion of these cytokines. Fc gamma receptor I antibodies significantly reversed CRP-mediated IL-10 inhibition. CRP significantly decreased intracellular cAMP, phospho-cAMP response element binding protein (pCREB), and adenyl cyclase activity. cAMP agonists reversed CRP-mediated IL-10 inhibition. Overexpression of wild-type and constitutively active CREB in THP-1 cells revealed attenuation of the inhibitory effect of CRP on LPS-induced IL-10 levels. CRP also inhibited hemoglobin:haptoglobin-induced IL-10 and heme oxygenase-1. Furthermore, administration of human CRP to rats significantly decreased IL-10 levels. CONCLUSIONS This study provides novel evidence that CRP, by decreasing IL-10 alters the antiinflammatory/proinflammatory balance, accentuating inflammation, which is pivotal in atherothrombosis.

Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis.

The cause of hepatic accumulation of fructose 1-phosphate on fructose loading.

1. The changes in the metabolite content in freeze-clamped livers of fed rats occurring on perfusion with 10mm-d-fructose have been examined. 2. The most striking effects of fructose were an accumulation of fructose 1-phosphate, as already known, up to 8.7mumol/g of liver within 10min, a loss of total adenine nucleotides (up to 35% after 40min) with a decrease in the ATP content to 23% within 10min, a sevenfold rise in the concentration of IMP to 1.1mumol/g and an eightfold rise of alpha-glycerophosphate to 1.1mumol/g. 3. There was a transient decrease in P(i) from 4.2 to 1.7mumol/g. Within 40min the P(i) content recovered to the normal value, probably because of an uptake of P(i) from the perfusion medium. 4. The degradation of the adenine nucleotides beyond the stage of AMP can be accounted for by the decrease of ATP and P(i). As ATP inhibits 5-nucleotidase, and as P(i) inhibits AMP deaminase any AMP arising in the tissue is liable to undergo dephosphorylation or deamination under the conditions occurring after fructose loading. 5. The content of lactate increased to 4.3mumol/g at 80min; pyruvate also increased and the [lactate]/[pyruvate] ratio remained within physiological limits. 6. The concentration of free fructose within the liver remained much below that in the perfusion medium, indicating that the rate of penetration of fructose into the tissue was lower than the rate of utilization. 7. The fission of fructose 1-phosphate by liver aldolase is inhibited by several phosphorylated intermediates, especially by IMP. This inhibition is competitive with a K(i) of 0.1mm. 8. The maximal rates of the enzymes synthesizing and splitting fructose 1-phosphate are about equal. The accumulation of fructose 1-phosphate on fructose loading is due to the inhibition of the fission of fructose 1-phosphate by the IMP arising from the degradation of the adenine nucleotides.

Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field

Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA.

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

Morin Attenuates Ovalbumin-Induced Airway Inflammation by Modulating Oxidative Stress-Responsive MAPK Signaling.

Asthma is one of the most common inflammatory diseases characterized by airway hyperresponsiveness, inflammation, and remodeling. Morin, an active ingredient obtained from Moraceae plants, has been demonstrated to have promising anti-inflammatory activities in a range of disorders. However, its impacts on pulmonary diseases, particularly on asthma, have not been clarified. This study was designed to investigate whether morin alleviates airway inflammation in chronic asthma with an emphasis on oxidative stress modulation. In vivo, ovalbumin- (OVA-) sensitized mice were administered with morin or dexamethasone before challenge. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained to perform cell counts, histological analysis, and enzyme-linked immunosorbent assay. In vitro, human bronchial epithelial cells (BECs) were challenged by tumor necrosis factor alpha (TNF-α). The supernatant was collected for the detection of the proinflammatory proteins, and the cells were collected for reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) evaluations. Severe inflammatory responses and remodeling were observed in the airways of the OVA-sensitized mice. Treatment with morin dramatically attenuated the extensive trafficking of inflammatory cells into the BALF and inhibited their infiltration around the respiratory tracts and vessels. Morin administration also significantly suppressed goblet cell hyperplasia and collagen deposition/fibrosis and dose-dependently inhibited the OVA-induced increases in IgE, TNF-α, interleukin- (IL-) 4, IL-13, matrix metalloproteinase-9, and malondialdehyde. In human BECs challenged by TNF-α, the levels of proteins such as eotaxin-1, monocyte chemoattractant protein-1, IL-8 and intercellular adhesion molecule-1, were consistently significantly decreased by morin. Western blotting and the 2',7'-dichlorofluorescein assay revealed that the increases in intracellular ROS and MAPK phosphorylation were abolished by morin, implying that ROS/MAPK signaling contributes to the relief of airway inflammation. Our findings indicate for the first time that morin alleviates airway inflammation in chronic asthma, which probably occurs via the oxidative stress-responsive MAPK pathway, highlighting a novel profile of morin as a potent agent for asthma management.

Enhanced waterlogging tolerance in barley by manipulation of expression of the N‐end rule pathway E3 ligase PROTEOLYSIS6

Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore, we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen-sensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

Platelet activation in type 2 diabetes mellitus.

The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia-induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox-sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro-thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high-risk non-diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long-term outcome in this high-risk population.

expression by oxidized linoleic

Hypercholesterolemia is associated with impairments in endothelium-dependent vascular relaxations. Paradoxically, endothelial production of nitrogen oxides is increased in early stages of hypercholesterolemia. Prior work has shown that oxidized low density lipoprotein (LDL) has both stimulatory and inhibitory effects on endothelial nitric oxide synthase expression (eNOS) and has focused on lysophosphatidyl choline (LPC) as a component of oxidized LDL which may modulate this effect. Another biologically active component of oxidized LDL is 13-hydroperoxyoctadecadienoic acid (13-HPODE), an oxidized form of linoleic acid. The purpose of this study was to determine the effect of HPODE on the expression of eNOS in bovine aortic endothelial cells (BAECs). Twenty four hour treatment of endothelial cells with HPODE caused a dose-dependent increase in eNOS mRNA levels as assessed by Northern analysis. The time response studies show that HPODE treatment significantly increased eNOS mRNA levels at 12 and 24 h. Concomitant with the increase in eNOS mRNA levels, 20 microM HPODE treatment significantly increased eNOS protein content and enzyme activity. Nuclear run-on studies indicated that the rate of transcription of eNOS gene was significantly elevated 4 h after HPODE treatment when compared to control cultures. Also, actinomycin D studies demonstrated that the half-life of eNOS mRNA was increased from 6 h to 12 h by HPODE treatment. Thus, HPODE-induced up-regulation of eNOS expression is mediated by both transcriptional and posttranscriptional mechanisms. These observations suggest that endothelial cells may attempt to compensate for oxidative injury by increasing expression of eNOS in early stages of hypercholesterolemia.

Involvement of ER Stress in Dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 Missense Mutations Shown by iPSC-Derived Oligodendrocytes

Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.

Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

Protection from olanzapine-induced metabolic toxicity in mice by acetaminophen and tetrahydroindenoindole

Objective:In mice and in humans, treatment with the second-generation antipsychotic drug olanzapine (OLZ) produces excessive weight gain, adiposity and secondary metabolic complications, including loss of glucose and insulin homeostasis. In mice consuming a high-fat (HF) diet, a similar phenotype develops, which is inhibited by the analgesic acetaminophen (APAP) and by the antioxidant tetrahydroindenoindole (THII). Therefore, we examined the ability of APAP and THII to prevent metabolic changes in mice receiving OLZ.Design and Measurement:C57BL/6J mice received either a normal diet or a HF diet, and were administered daily dosages of OLZ (3 mg kg−1 body weight), alone or with APAP (30 mg kg−1 body weight) or THII (4.5 mg kg−1 body weight), for 10 weeks. Parameters of body composition and metabolism, including glucose and insulin homeostasis and oxidative stress, were examined. Results:OLZ treatment doubled the HF diet-induced increases in body weight and percent body fat. These increases were partially prevented by both APAP and THII, although food consumption was constant in all groups. The THII protection was associated with an increase in whole body and mitochondrial respiration. OLZ also exacerbated, and both APAP and THII prevented, HF diet-induced loss of glucose tolerance and insulin resistance. As increased body fat promotes insulin resistance by a pathway involving oxidative stress, we evaluated production of reactive oxygen and lipid peroxidation in white adipose tissue (WAT). HF diet caused an increase in lipid peroxidation, NADPH-dependent O2 uptake and H2O2 production, which were further exacerbated by OLZ. APAP, THII and the NADPH oxidase inhibitor, diphenyleneiodonium chloride, each abolished oxidative stress in WAT.Conclusions:We conclude that both APAP and THII intervene in the development of obesity and metabolic complications associated with OLZ treatment.

Liraglutide improves hippocampal synaptic plasticity associated with increased expression of Mash1 in ob/ob mice

Objective:Consumption of high-fat diet exerts adverse effects on learning and memory formation, which is linked to impaired hippocampal function. Activation of glucagon-like peptide-1 (GLP-1) signalling ameliorates detrimental effects of obesity-diabetes on cognitive function; however, mechanisms underlying these beneficial actions remain unclear. This study examined effects of daily subcutaneous treatment with GLP-1 mimetic, Liraglutide, on synaptic plasticity, hippocampal gene expression and metabolic control in adult obese diabetic (ob/ob) mice. Results:Long-term potentiation (LTP) induced by area CA1 was completely abolished in ob/ob mice compared with lean controls. Deleterious effects on LTP were rescued (P<0.001) with Liraglutide. Indeed, Liraglutide-treated mice exhibited superior LTP profile compared with lean controls (P<0.01). Expression of hippocampal brain-derived neurotropic factor and neurotrophic tyrosine kinase receptor-type 2 were not significantly different, but synaptophysin and Mash1 were decreased in ob/ob mice. Treatment with Liraglutide over 21 days increased expression of Mash1 in ob/ob mice (2.0-fold; P<0.01). These changes were associated with significantly reduced plasma glucose (21% reduction; P<0.05) and markedly improved plasma insulin concentrations (2.1- to 3.3-fold; P<0.05 to P<0.01). Liraglutide also significantly reduced the glycaemic excursion following an intraperitonal glucose load (area under curve (AUC) values: 22%; P<0.05) and markedly enhanced the insulin response to glucose (AUC values: 1.6-fold; P<0.05). O2 consumption, CO2 production, respiratory exchange ratio and energy expenditure were not altered by Liraglutide therapy. On day 21, accumulated food intake (32% reduction; P<0.05) and number of feeding bouts (32% reduction; P<0.05) were significantly reduced but simple energy restriction was not responsible for the beneficial actions of Liraglutide. Conclusion:Liraglutide elicits beneficial effects on metabolic control and synaptic plasticity in mice with severe obesity and insulin resistance mediated in part through increased expression of Mash1 believed to improve hippocampal neurogenesis and cell survival.

Angiopoietin-2 is increased in sepsis and inversely associated with nitric oxide-dependent microvascular reactivity

INTRODUCTION Angiopoietin-2 (ang-2), an angiogenic peptide released by endothelial cell Weibel-Palade bodies (WPBs), increases endothelial activation and vascular permeability. Ang-2 is raised in severe sepsis but the mechanisms underlying this are not known. Nitric oxide (NO) inhibits WPB exocytosis, and bioavailability of endothelial NO is decreased in sepsis. We hypothesized that endothelial NO bioavailability would be inversely correlated with ang-2 concentrations in sepsis. METHODS Plasma ang-2, vascular endothelial growth factor (VEGF) and endothelial-active cytokines were assessed in 83 patients with early sepsis and 41 hospital controls, and related to reactive hyperaemia-peripheral arterial tonometry, RH-PAT, a measure of endothelial NO bioavailability. RESULTS Plasma Ang-2 was elevated in sepsis (median [interquartile range (IQR)], ng/ml: severe sepsis 12.4 [8.5-33.4], sepsis without organ failure 6.1 [5.0-10.4], controls 2.7 [2.2-3.6], P < 0.0001). It correlated inversely with RH-PAT (r = -0.38, P < 0.0001) and positively with IL-6 (r = 0.57, P < 0.0001) and degree of organ failure (sequential organ function assessment score) (r = 0.58, P < 0.0001). The correlation of ang-2 with RH-PAT persisted after controlling for sepsis severity. In a longitudinal mixed-effects model, recovery of RH-PAT over time was associated with decline in ang-2. CONCLUSIONS Ang-2 is elevated in proportion to sepsis severity, and inversely correlated with NO-dependent microvascular reactivity. Impaired endothelial NO bioavailability may contribute to increased endothelial cell release of ang-2, endothelial activation and capillary leak. Agents that increase endothelial NO bioavailability or inhibit WPB exocytosis and/or Ang-2 activity may have therapeutic potential in sepsis.

Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Analysis of human cytomegalovirus-encoded microRNA activity during infection.

MicroRNAs (miRNAs) are expressed in a wide variety of organisms, ranging from plants to animals, and are key posttranscriptional regulators of gene expression. Virally encoded miRNAs are unique in that they could potentially target both viral and host genes. Indeed, we have previously demonstrated that a human cytomegalovirus (HCMV)-encoded miRNA, miR-UL112, downregulates the expression of a host immune gene, MICB. Remarkably, it was shown that the same miRNA also downregulates immediate-early viral genes and that its ectopic expression resulted in reduced viral replication and viral titers. The targets for most of the viral miRNAs, and hence their functions, are still unknown. Here we demonstrate that miR-UL112 also targets the UL114 gene, and we present evidence that the reduction of UL114 by miR-UL112 reduces its activity as uracil DNA glycosylase but only minimally affects virus growth. In addition, we show that two additional HCMV-encoded miRNAs, miR-US25-1 and miR-US25-2, reduce the viral replication and DNA synthesis not only of HCMV but also of other viruses, suggesting that these two miRNAs target cellular genes that are essential for virus growth. Thus, we suggest that in addition to miR-UL112, two additional HCMV miRNAs control the life cycle of the virus.

Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence

The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans' replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal's effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells were used for infection, which confirmed target specificity and ruled out non-specific effects of the drugs on the Galleria host. Thus, this study suggests that RLS modulating drugs, such as Sir2p agonists, shift lifespan and vulnerability of the fungal population, and should be further investigated as a potential class of novel antifungal drug targets that can enhance antifungal efficacy.

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.

Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.

The Ran GTPase regulates mitotic spindle assembly

Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.

Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice

Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.

Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene.

One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of β-oxidation flux by labeled palmitate demonstrates that tBid inhibits β-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on β-oxidation. The unexpected role of tBid in the regulation of lipid β-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

Keeping your senescent cells under control

Cellular senescence is a stable form of cell-cycle arrest which is thought to limit the proliferative potential of premalignant cells [1]. The senescence phenotype was initially described by Hayflick and Moorhead in 1961 on human fibroblasts undergoing replicative exhaustion in culture [2]. It has been shown that senescence can be triggered in different cell types in response to diverse forms of cellular damage or stress (for review see [1]). Importantly, while senescence was denounced as a tissue culture phenomenon for many years, recent in vivo studies demonstrated that cellular senescence represents a potent failsafe mechanism against tumorigenesis and contributes to the cytotoxicity of certain anticancer agents (see for example [3-7]). Interestingly, senescent cells have also been observed in certain aged or damaged tissues and there is growing evidence that senescence checkpoints can affect the regenerative reserve of tissues and organismal aging [8-11]. However, senescence may also have positive effects on organ maintenance by limiting pathological responses to acute forms of injury such as fibrotic scarring in response to chemical induced liver injury [12]. Over the past years it was also shown that senescent cells can communicate with their environment by secreting a myriad of cytokines and growth factors. Interestingly, this "senescence associated secretory phenotype (SASP)" seems to be a double edged sword regarding tumor initiation and maintenance:    i)    On the one hand, it has been shown that the SASP can have pro-tumorigenic effects. In an experimental system it was shown that senescent mesenchymal cells can enhance the tumorigenicity of surrounding breast cancer cells [13]. ii)    Similarly, it is possible that the SASP enhances selection of transformed cell clones in aged organ systems. It has been shown that loss of proliferative competition of non-transformed cells can accelerate leukemogenesis [14]. It remains to be seen whether aberrant secretion of cytokines and growth factors by the SASP can accelerated this process in aged and chronically damage organ systems. iii)    In contrast to its pro-tumorigenic aspect, the SASP could also have anti-tumor effects. A recent study showed that in a mosaic liver cancer mouse model the activation of p53 induced senescence, an upregulation of inflammatory cytokines, and activation of innate immune responses leading to tumour cell clearance [15]. iv)    In further support that the SASP could have anti-tumor activities, a series of recent papers showed that components of the SASP can stabilize the senescence cell cycle arrest via an autoregulatory feedback loop [16,17] or induces apoptosis of tumor cells [18]. In addition to its effects on tumorigenesis, the SASP could also influence tissue aging. Studies on aging telomere dysfunctional mice have provided direct experimental evidence for an in vivo activation of the SASP in response to telomere dysfunction [19]. Interestingly, this in vivo SASP provoked alterations in stem cell differentiation (skewing of hematopoiesis towards reduction in lymphopoiesis and enhancement of myelopoiesis) that are also characteristic signs of human aging. Figure 1. Different cellular stresses can induce senescence including telomere shortening, DNA damage, and oncogene activation. Senescence of tumor cells ... In light of the many possible roles o the SASP in aging and carcinogenesis, it appears to be of utmost importance to decipher regulatory pathways controlling the SASP. In a current publication, Bhaumik et al. have identified 2 microRNAs (miR-146a/b) that negatively regulate the secretion of IL-6 and IL-8 - two of the SASP [20]. The authors show that these microRNAs are up-regulated at late stages of senescence, many days after a permanent cell cycle arrest has been established. Interestingly, the inhibitory miRs are most strongly up-regulated in senescence of cell lines that show a strong SASP but not in cell lines characterized by a weak SASP. The authors propose a new concept indicating that miRs 146a and b function in a negative feedback loop preventing an over-activation of the SASP in senescent cells. The authors present some initial data suggesting that activation of this negative feedback loop involves IL-1 receptor, IRAK-1, and NFκB signalling leading to an up-regulation of miRs-146a and b. A direct proof that this proposed feedback loop suppresses over-activation of the SASP remains to be demonstrated in future studies. The authors show that blockage of IL-1-receptor signalling prevents both the up-regulation of miRs-146a and b as well as Il-6 secretion. To confirm their new concept, it would be important to show that a selective blockage of miRs-146a and b results in over-activation of the SASP. The work by Bhaumik et al. places mir-146a/b as central players to control IL-6 and IL-8 expression within the SASP. MicroRNAs are emerging therapeutic targets because their expression levels can be effectively modulated via the use of antagomirs (see for example [21]). Also, for increasing microRNA expression, microRNAs can be delivered into cellsin vivo (see for example [22]). Therefore, it will be interesting to functionally test the impact of mir-146 inhibition on tumorigenesis and aging in relevant mouse models. Such studies will be of particular interest, as recent work showed that IL-6 secretion by senescent cells is relevant for initiating and maintaining the senescene response via an autocrine loop [17]. A reduction of miR-146 could increase IL-6 levels in senescent cells, which should stabilize the senescence program and reduce the risk of malignant transformation. Furthermore, it can be speculated that reduction of mir-146 a/b will increase NfκB activation via IRAK1. As NfκB is modulating the expression of various inflammation associated genes, this may also lead to increased clearance of senescent tumor cells by the innate immune system. However, it should be mentioned that Il-6 secreted by senescent cells can also act as a mitogen for surrounding cells, thus potentially increasing the risk of malignant transformation [13,17]. Besides its function in SASP modulation, miR-146 was also reported to target the mRNAs of the BRCA1 and BRCA2 tumor suppressors. In a recent study a G to C polymorphism in miR-146, which leads to an increased processing and release of the mature microRNA, can predict an early onset of breast cancer [23]. Taken together, the study of Bhaumik et al. opens an interesting new research area dealing with the gene regulatory mechanisms that control activation of the SASP. Given the diverse roles of the SASP in modulating tumor progression, immune surveillance of damaged cells, and the stabilization of the senescence arrest itself, it will be of great interest to analyse the influence of SASP regulatory pathways during aging and cancer.

Periostin Deficiency Increases Bone Damage and Impairs Injury Response to Fatigue Loading in Adult Mice

Bone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.

Green tea polyphenol causes differential oxidative environments in tumor versus normal epithelial cells.

Green tea polyphenols (GTPPs) are considered beneficial to human health, especially as chemopreventive agents. Recently, cytotoxic reactive oxygen species (ROS) were identified in tumor and certain normal cell cultures incubated with high concentrations of the most abundant GTPP, (-)-epigallocatechin-3-gallate (EGCG). If EGCG also provokes the production of ROS in normal epithelial cells, it may preclude the topical use of EGCG at higher doses. The current study examined the oxidative status of normal epithelial, normal salivary gland, and oral carcinoma cells treated with EGCG, using ROS measurement and catalase and superoxide dismutase activity assays. The results demonstrated that high concentrations of EGCG induced oxidative stress only in tumor cells. In contrast, EGCG reduced ROS in normal cells to background levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromodeoxyuridine incorporation data were also compared between the two oral carcinoma cell lines treated by EGCG, which suggest that a difference in the levels of endogenous catalase activity may play an important role in reducing oxidative stress provoked by EGCG in tumor cells. It is concluded that pathways activated by GTPPs or EGCG in normal epithelial versus tumor cells create different oxidative environments, favoring either normal cell survival or tumor cell destruction. This finding may lead to applications of naturally occurring polyphenols to enhance the effectiveness of chemo/radiation therapy to promote cancer cell death while protecting normal cells.

Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis.

Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

OBJECTIVE To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.

VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation.

BACKGROUND Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. OBJECTIVE To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. METHODS Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. RESULTS Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. CONCLUSION Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.

Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

Hepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2 alpha and attenuates ISG protein expression despite normal ISG mRNA induction. ISG protein induction is restored and the antiviral effects of interferon are enhanced when PKR expression is suppressed in interferon-treated infected cells. Whereas host protein translation, including antiviral ISGs, is suppressed by activated PKR, HCV IRES-dependent translation is not. These results suggest that the ability of HCV to activate PKR may, paradoxically, be advantageous for the virus during an IFN response by preferentially suppressing the translation of ISGs.

Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2

Rett syndrome is caused by loss-of-function mutations in the gene encoding the methyl DNA-binding factor MeCP2. As brain mass and neuronal complexity tend to be diminished in Rett patients, we tested whether MeCP2 directly influences the morphological complexity of developing neurons. Our results show that cultured mouse neurons overexpressing MeCP2beta (MECP2A) develop more complex morphologies, having longer axonal and dendritic processes, and an increased number of axonal and dendritic terminal endings. We then tested whether overexpressing a mutant form of MeCP2beta lacking its carboxyl terminus would elicit the same effects. Interestingly, while neurons overexpressing this mutant failed to enhance axonal and dendritic process elongation, the complexity of their axonal and dendritic processes remained significantly elevated. Taken together, these data support the hypothesis that MeCP2 directly regulates neuronal maturation and/or synaptogenesis, and provides evidence that MeCP2 may influence neuritic elongation and process branching through different mechanisms.

Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD.

BACKGROUND Interleukin (IL)-17F, produced in IL-23R-expressing Th17 cells, is a novel member of the IL-17 cytokine family. Given the association of IL23R with inflammatory bowel disease (IBD), we characterized the role of IL-17F in IBD including its intestinal gene expression and the effect of the IL17F p. His161Arg polymorphism on disease susceptibility and phenotype of Crohn's disease (CD) and ulcerative colitis (UC). In addition, we analyzed the IL17F p. His161Arg polymorphism for potential epistasis with IL23R and NOD2/CARD15 variants. METHODS Intestinal IL-17F mRNA expression was measured by quantitative polymerase chain reaction (PCR). Genomic DNA from 1682 individuals (CD: n = 499; UC: n = 216; controls: n = 967) was analyzed for the presence of the IL17F p. His161Arg polymorphism, the 3 NOD2 variants, p. Arg702Trp, p. Gly908Arg, and p. Leu1007fsX1008, and 10 CD-associated IL23R variants. RESULTS Intestinal IL-17F mRNA expression was 4.4-fold increased in inflamed colonic lesions compared to uninflamed biopsies in CD (P = 0.016) but not in UC. However, the mean intestinal IL-17F mRNA expression was higher in UC than in CD (P < 0.0001). The IL17F p. His161Arg substitution was observed with similar frequencies in IBD patients and controls and was not associated with a certain disease phenotype, but weakly associated with a low body mass index (BMI; P = 0.009) and an earlier age of disease onset (P = 0.039) in UC. There was no evidence for epistasis between the IL17F p. His161Arg polymorphism and IBD-associated single nucleotide polymorphisms within the IL23R gene. CONCLUSIONS Intestinal IL17F gene expression is increased in active CD. The IL17F p. His161Arg polymorphism is not associated with IBD susceptibility and has no epistatic interaction with CD-associated IL23R variants.

Simian immunodeficiency virus–induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor–deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells.

Hydrogen sulfide (H(2)S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H(2)S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H(2)S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H(2)S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H(2)S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H(2)S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function.

Killing by bactericidal antibiotics does not depend on reactive oxygen species.

Bactericidal antibiotics kill by modulating their respective targets. This traditional view has been challenged by studies that propose an alternative, unified mechanism of killing, whereby toxic reactive oxygen species (ROS) are produced in the presence of antibiotics. We found no correlation between an individual cell's probability of survival in the presence of antibiotic and its level of ROS. An ROS quencher, thiourea, protected cells from antibiotics present at low concentrations, but the effect was observed under anaerobic conditions as well. There was essentially no difference in survival of bacteria treated with various antibiotics under aerobic or anaerobic conditions. This suggests that ROS do not play a role in killing of bacterial pathogens by antibiotics.

Disturbed sleep in bipolar disorder is related to an elevation of IL-6 in peripheral monocytes.

Bipolar disorder is a severe psychiatric disorder that is associated with persistent changes in the quality, duration and architecture of sleep. Currently there is no unifying hypothesis explaining the alterations in sleep observable in patients with bipolar disorder and management is often difficult though vital. Sleep is modified by various cytokines including IL-6. Elevated levels of IL-6 are associated with a poorer quality of sleep and changes in the architecture of sleep similar to those observed in bipolar disorder. Therapeutic administration of Interferon causes elevations of intrathecal IL-6 concentrations and appears to provoke a deteriorating quality of sleep. The blockade of IL-6 with tocilizumab in rheumatoid arthritis is associated with improvements in the quality of sleep. Bipolar disorder is associated with elevated levels of IL-6 and in particular elevated levels of mRNA coding for IL-6 in peripheral monocytes. We propose that the changes observed in the sleep of patients with bipolar disorder are related to the elevation of IL-6 and that this correlates with an elevated expression of mRNA coding for IL-6 expression in peripheral monocytes.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC.

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator (agr) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans-autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.

Antioxidants attenuate the plasma cytokine response to exercise in humans.

Exercise increases plasma TNF-alpha, IL-1beta, and IL-6, yet the stimuli and sources of TNF-alpha and IL-1beta remain largely unknown. We tested the role of oxidative stress and the potential contribution of monocytes in this cytokine (especially IL-1beta) response in previously untrained individuals. Six healthy nonathletes performed two 45-min bicycle exercise sessions at 70% of Vo(2 max) before and after a combination of antioxidants (vitamins E, A, and C for 60 days; allopurinol for 15 days; and N-acetylcysteine for 3 days). Blood was drawn at baseline, end-exercise, and 30 and 120 min postexercise. Plasma cytokines were determined by ELISA and monocyte intracellular cytokine level by flow cytometry. Before antioxidants, TNF-alpha increased by 60%, IL-1beta by threefold, and IL-6 by sixfold secondary to exercise (P < 0.05). After antioxidants, plasma IL-1beta became undetectable, the TNF-alpha response to exercise was abolished, and the IL-6 response was significantly blunted (P < 0.05). Exercise did not increase the percentage of monocytes producing the cytokines or their mean fluorescence intensity. We conclude that in untrained humans oxidative stress is a major stimulus for exercise-induced cytokine production and that monocytes play no role in this process.

Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line.

Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.