4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.     

Randomized, controlled trial of therapy interruption in chronic HIV-1 infection

Background

Approaches to limiting exposure to antiretroviral therapy (ART) drugs are an active area of HIV therapy research. Here we present longitudinal follow-up of a randomized, open-label, single-center study of the immune, viral, and safety outcomes of structured therapy interruptions (TIs) in patients with chronically suppressed HIV-1 infection as compared to equal follow-up of patients on continuous therapy and including a final therapy interruption in both arms.

Methods and Findings

Forty-two chronically HIV-infected patients on suppressive ART with CD4 counts higher than 400 were randomized 1:1 to either (1) three successive fixed TIs of 2, 4, and 6 wk, with intervening resumption of therapy with resuppression for 4 wk before subsequent interruption, or (2) 40 wk of continuous therapy, with a final open-ended TI in both treatment groups. Main outcome was analysis of the time to viral rebound (>5,000 copies/ml) during the open-ended TI. Secondary outcomes included study-defined safety criteria, viral resistance, therapy failure, and retention of immune reconstitution.There was no difference between the groups in time to viral rebound during the open-ended TI (continuous therapy/single TI, median [interquartile range] = 4 [1–8] wk, n = 21; repeated TI, median [interquartile range] = 5 [4–8] wk, n = 21; p = 0.36). No differences in study-related adverse events, viral set point at 12 or 20 wk of open-ended interruption, viral resistance or therapy failure, retention of CD4 T cell numbers on ART, or retention of lymphoproliferative recall antigen responses were noted between groups. Importantly, resistance detected shortly after initial viremia following the open-ended TI did not result in a lack of resuppression to less than 50 copies/ml after reinitiation of the same drug regimen.

Conclusion

Cycles of 2- to 6-wk time-fixed TIs in patients with suppressed HIV infection failed to confer a clinically significant benefit with regard to viral suppression off ART. Also, secondary analysis showed no difference between the two strategies in terms of safety, retention of immune reconstitution, and clinical therapy failure. Based on these findings, we suggest that further clinical research on the long-term consequences of TI strategies to decrease drug exposure is warranted.     

Analysis of expansion of myeloid progenitors in mice to identify leukemic susceptibility genes

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.     

Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway

In vitro studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis in vivo remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and jnk1-/- mice but markedly decreased in jnk2-/- mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of jnk2, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in jnk2-/- mice Bid cleavage and mitochondrial translocation and cytochrome c release were markedly decreased. This effect was secondary to the failure of jnk2-/- mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in jnk2 null mice after GalN/TNF treatment. Ablation of jnk2 did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in jnk1-/- mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.     

A nitrate-induced frq-less oscillator in Neurospora crassa

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.     

Iron deposition and increased alveolar septal capillary density in nonfibrotic lung tissue are associated with pulmonary hypertension in idiopathic pulmonary fibrosis

Background

Early diagnosis of pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) has potential prognostic and therapeutic implications but can be difficult due to the lack of specific clinical manifestations or accurate non-invasive tests. Histopathologic parameters correlating with PH in IPF are also not known. Remodeling of postcapillary pulmonary vessels has been reported in the nonfibrotic areas of explanted lungs from IPF patients. We hypothesized that iron deposition and increased alveolar capillaries, the findings often seen in postcapillary PH, might predict the presence of clinical PH, independent of the severity of fibrosis or ventilatory dysfunction in IPF patients. To test this hypothesis, we examined the association between these histologic parameters and the degree of PH, with consideration of the severity of disease in IPF.

Methods

Iron deposition and alveolar septal capillary density (ASCD) were evaluated on histologic sections with hematoxylin-eosin, iron, elastin and CD34 stainings. Percentage of predicted forced vital capacity (FVC%) was used for grading pulmonary function status. Fibrosis score assessed on high resolution computed tomography (HRCT) was used for evaluating overall degree of fibrosis in whole lungs. Right ventricular systolic pressure (RVSP) by transthoracic echocardiography was used for the estimation of PH. Univariate and multivariate regression analyses were performed.

Results

A cohort of 154 patients was studied who had the clinicopathological diagnosis of IPF with surgical lung biopsies or explants during the period of 1997 to 2006 at Mayo Clinic Rochester. In univariate analysis, RVSP in our IPF cases was associated with both iron deposition and ASCD (p < 0.001). In multivariate analysis with FVC% and HRCT fibrosis score included, iron deposition (p = 0.02), but not ASCD (p = 0.076), maintained statistically significant association with RVSP. FVC% was associated with RVSP on univariate analysis but not on multivariate analysis, while fibrosis score lacked any association with RVSP by either univariate or multivariate analyses.

Conclusions

Iron deposition and ASCD in non fibrotic lung tissue showed an association with RVSP, suggesting that these features are possible morphologic predictors of PH in IPF.     

Habitat degradation affects the summer activity of polar bears

Understanding behavioral responses of species to environmental change is critical to forecasting population-level effects. Although climate change is significantly impacting species’ distributions, few studies have examined associated changes in behavior. Polar bear (Ursus maritimus) subpopulations have varied in their near-term responses to sea ice decline. We examined behavioral responses of two adjacent subpopulations to changes in habitat availability during the annual sea ice minimum using activity data. Location and activity sensor data collected from 1989 to 2014 for 202 adult female polar bears in the Southern Beaufort Sea (SB) and Chukchi Sea (CS) subpopulations were used to compare activity in three habitat types varying in prey availability: (1) land; (2) ice over shallow, biologically productive waters; and (3) ice over deeper, less productive waters. Bears varied activity across and within habitats with the highest activity at 50–75% sea ice concentration over shallow waters. On land, SB bears exhibited variable but relatively high activity associated with the use of subsistence-harvested bowhead whale carcasses, whereas CS bears exhibited low activity consistent with minimal feeding. Both subpopulations had fewer observations in their preferred shallow-water sea ice habitats in recent years, corresponding with declines in availability of this substrate. The substantially higher use of marginal habitats by SB bears is an additional mechanism potentially explaining why this subpopulation has experienced negative effects of sea ice loss compared to the still-productive CS subpopulation. Variability in activity among, and within, habitats suggests that bears alter their behavior in response to habitat conditions, presumably in an attempt to balance prey availability with energy costs.