Gene targeting of the cysteine peptidase cathepsin H impairs lung surfactant in mice

Background

The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed.

Methodology/Principal Findings

Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh ^−/− mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh ^−/− mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh^+/+ and Ctsh ^−/− mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh ^−/− mice.

Conclusions/Significance

We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.     

Loss of DJ-1 does not affect mitochondrial respiration but increases ROS production and mitochondrial permeability transition pore opening

Background

Loss of function mutations in the DJ-1 gene have been linked to recessively inherited forms of Parkinsonism. Mitochondrial dysfunction and increased oxidative stress are thought to be key events in the pathogenesis of Parkinson’s disease. Although it has been reported that DJ-1 serves as scavenger for reactive oxidative species (ROS) by oxidation on its cysteine residues, how loss of DJ-1 affects mitochondrial function is less clear.

Methodology/Principal Findings

Using primary mouse embryonic fibroblasts (MEFs) or brains from DJ-1−/− mice, we found that loss of DJ-1 does not affect mitochondrial respiration. Specifically, endogenous respiratory activity as well as basal and maximal respiration are normal in intact DJ-1−/− MEFs, and substrate-specific state 3 and state 4 mitochondrial respiration are also unaffected in permeabilized DJ-1−/− MEFs and in isolated mitochondria from the cerebral cortex of DJ-1−/− mice at 3 months or 2 years of age. Expression levels and activities of all individual complexes composing the electron transport system are unchanged, but ATP production is reduced in DJ-1−/− MEFs. Mitochondrial transmembrane potential is decreased in the absence of DJ-1. Furthermore, mitochondrial permeability transition pore opening is increased, whereas mitochondrial calcium levels are unchanged in DJ-1−/− cells. Consistent with earlier reports, production of reactive oxygen species (ROS) is increased, though levels of antioxidative enzymes are unaltered. Interestingly, the decreased mitochondrial transmembrane potential and the increased mitochondrial permeability transition pore opening in DJ-1−/− MEFs can be restored by antioxidant treatment, whereas oxidative stress inducers have the opposite effects on mitochondrial transmembrane potential and mitochondrial permeability transition pore opening.

Conclusions/Significance

Our study shows that loss of DJ-1 does not affect mitochondrial respiration or mitochondrial calcium levels but increases ROS production, leading to elevated mitochondrial permeability transition pore opening and reduced mitochondrial transmembrane potential.     

The Mercaptopyruvate Sulfurtransferase of Trichomonas vaginalis Links Cysteine Catabolism to the Production of Thioredoxin Persulfide

Trichomonas vaginalis is a protozoan parasite of humans that is able to synthesize cysteine de novo using cysteine synthase but does not produce glutathione. In this study, high pressure liquid chromatography analysis confirmed that cysteine is the major intracellular redox buffer by showing that T. vaginalis contains high levels of cysteine (∼600 μm) comprising more than 70% of the total thiols detected. To investigate possible mechanisms for the regulation of cysteine levels in T. vaginalis, we have characterized enzymes of the mercaptopyruvate pathway. This consists of an aspartate aminotransferase (TvAspAT1), which transaminates cysteine to form 3-mercaptopyruvate (3-MP), and mercaptopyruvate sulfurtransferase (TvMST), which transfers the sulfur of 3-MP to a nucleophilic acceptor, generating pyruvate. TvMST has high activity with 3-MP as a sulfur donor and can use several thiol compounds as sulfur acceptor substrates. Our analysis indicated that TvMST has a k_cat/K_m for reduced thioredoxin of 6.2 × 10⁷ m⁻¹ s⁻¹, more than 100-fold higher than that observed for β-mercaptoethanol and cysteine, suggesting that thioredoxin is a preferred substrate for TvMST. Thiol trapping and mass spectrometry provided direct evidence for the formation of thioredoxin persulfide as a product of this reaction. The thioredoxin persulfide could serve a biological function such as the transfer of the persulfide to a target protein or the sequestered release of sulfide for biosynthesis. Changes in MST activity of T. vaginalis in response to variation in the supply of exogenous cysteine are suggestive of a role for the mercaptopyruvate pathway in the removal of excess intracellular cysteine, redox homeostasis, and antioxidant defense.     

Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes

Background

Two pertussis toxin sensitive G_i proteins, G_i2 and G_i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G_i isoforms are functionally distinct. To test for isoform-specific functions of G_i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).

Methods

Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα_i2 (Gα_i2 ^−/−) or Gα_i3 (Gα_i3 ^−/−). mRNA levels of Gα_i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα_i and Ca_vα₁ protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.

Results

In cardiac tissue from Gα_i2 ^−/− mice, Gα_i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gα_i3 ^−/− mouse hearts, Gα_i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα_i2 ^−/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα_i3 ^−/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα_i2 (but not of Gα_i3) and following treatment with pertussis toxin in Gα_i3 ^−/−. The pore forming Ca_vα₁ protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca_vα₁ and Ca_vβ₂ subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα_i2.

Conclusion

Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα_i proteins. In particular, loss of Gα_i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.     

Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice

Background

Lysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm.

Methodology/Principal Findings

Affymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G⁺ myeloid lineage cells of LAL knock-out (lal−/−) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal−/− Ly6G⁺ myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal−/− Ly6G⁺ myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells.

Conclusions/Significance

This comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal−/− mouse model.     

A comprehensive evaluation of potential lung function associated genes in the SpiroMeta general population sample

Rationale

Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium).

Objectives

To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample.

Methods

We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations.

Results

The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV₁ or FEV₁/FVC traits using a carefully defined significance threshold of 1.3×10⁻⁵. The most significant loci associated with FEV₁ include SNPs tagging MACROD2 (P = 6.81×10⁻⁵), CNTN5 (P = 4.37×10⁻⁴), and TRPV4 (P = 1.58×10⁻³). Among ever-smokers, SERPINA1 showed the most significant association with FEV₁ (P = 8.41×10⁻⁵), followed by PDE4D (P = 1.22×10⁻⁴). The strongest association with FEV₁/FVC ratio was observed with ABCC1 (P = 4.38×10⁻⁴), and ESR1 (P = 5.42×10⁻⁴) among ever-smokers.

Conclusions

Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV₁ among smokers in the general population.     

Direct sensing of endothelial oxidants by vascular endothelial growth factor receptor-2 and c-Src

Background

ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis ‘senses’ NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs.

Methodology/Principal Findings

Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47^phox subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS.

Conclusion/Significance

These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can ‘sense’ redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.     

Cathepsin S deficiency results in abnormal accumulation of autophagosomes in macrophages and enhances Ang II-induced cardiac inflammation

Background

Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice.

Methods and Results

Cat S-knockout (Cat S^−/−) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S^−/− mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor β and interleukin 1β) were significantly greater in Cat S^−/− than WT hearts. These Ang II-induced effects in Cat S^−/− mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages.

Conclusion

Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis.     

Influences of chloride and hypochlorite on neutrophil extracellular trap formation

Background

The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl⁻) and hydrogen peroxide (H₂O₂), and its product hypochlorite (HOCl) in NETosis.

Findings

In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl⁻, a substrate for MPO, also reduced NETosis. While exogenous addition of H₂O₂ and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils.

Conclusions

Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl⁻ homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.     

Cysteinyl leukotriene signaling aggravates myocardial hypoxia in experimental atherosclerotic heart disease

Background

Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe^−/− mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia.

Methods and principal findings

Myocardial biopsies from Apoe^−/− mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C₄ synthase (LTC₄S) and CysLT1 expression in the myocardium of Apoe^−/− mice. Acute bouts of hypoxia further induced LTC₄S expression, increased LTC₄S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe^−/− mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC₄S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling.

Conclusion

Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.     

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery

Background

The rate-limiting step that determines the dominant time constant (τ_D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca²⁺-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ_D is modulated by background light in mouse rods.

Methodology/Principal Findings

We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca²⁺ shortens the dominant time constant (τ_D) of saturated photoresponse recovery, i.e., when extracellular free Ca²⁺ is decreased from 1 mM to ∼25 nM, the τ_D is reversibly increased ∼1.5–2-fold.

Conclusions

We conclude that the increase in τ_D during low Ca²⁺ treatment is not due to increased [cGMP], increased [Na⁺] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca²⁺-dependent mechanism controls the life time of active PDE.     

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels

Background and Aims

Experimental evidence in the literature suggests that O₂^•− produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O₂^•−.

Methods

Stress treatments were imposed using 150 mm NaCl or 300 mm sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O₂^•− was determined using nitro blue tetrazolium, and H₂O₂ was determined using 2′, 7′-dichlorofluorescin.

Key Results

In non-stressed plants, the distribution of accelerating growth and highest O₂^•− levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O₂^•− levels increased in sorbitol-treated roots and decreased in NaCl-treated roots.

Conclusions

The lack of association between apoplastic O₂^•− levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O₂^•− may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.Key words: Root tip growth, Zea mays, salt stress, reactive oxygen species, ROS     

Functional characterization of the octenol receptor neuron on the maxillary palps of the yellow fever mosquito, Aedes aegypti

Background

1-Octen-3-ol (octenol) is a common attractant released by vertebrates which in combination with carbon dioxide (CO₂) attracts hematophagous arthropods including mosquitoes. A receptor neuron contained within basiconic sensilla on the maxillary palps of adult mosquitoes responds selectively to 1-octen-3-ol. Recently, an odorant receptor (AaegOR8) known to occur on the maxillary palps was expressed in a heterologous system and demonstrated to be selectively sensitive to (R)-(−)-1-octen-3-ol, one of two enantiomeric forms. Lesser responses were elicited by stimulation with the (S)-enantiomer and various structural analogs.

Methodology/Principal Findings

Here we characterize the specificity of the octenol receptor neuron in the yellow fever mosquito, Aedes aegypti (L.), in vivo using single cell recordings. The octenol neuron is exquisitely sensitive to (R)-(−)-1-octen-3-ol; comparable responses to (S)-(+)-1-octen-3-ol were elicited only at stimulus doses over 100× that required for the (R)-enantiomer. An intermediate response closer to that elicited by the (R)-(−)-enantiomer was elicited by racemic 1-octen-3-ol. Small structural changes in (R)-(−)-1-octen-3-ol resulted in large decreases in responses. Increases in spike activity were also elicited in the octenol neuron by 2-undecanone, a known repellent; other repellents (DEET, IR3535 and picaridin) were inactive.

Conclusions/Significance

The results of our electrophysiological studies of the octenol receptor neuron in vivo approximates results of a previous study of the octenol receptor (AaegOR8 with its obligate partner Aaeg\ORco) expressed heterologously in Xenopus oocytes. By comparison of our current results with those of the heterologous expression study, we conclude that specificity of the octenol receptor neuron can be explained largely by characteristics of the OR alone without other associated proteins present in vivo. Our findings show that repellents may have specific stimulatory effects on receptor neurons and support the notion of repellents as modulators of mosquito odorant receptor activity.     

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression

Background

Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish.

Methodology/Principal Findings

Individual zebrafish (n = 10) were able to swim at a critical swimming speed (U_crit) of 0.548±0.007 m s⁻¹ or 18.0 standard body lengths (BL) s⁻¹. The optimal swimming speed (U_opt) at which energetic efficiency is highest was 0.396±0.019 m s⁻¹ (13.0 BL s⁻¹) corresponding to 72.26±0.29% of U_crit. The cost of transport at optimal swimming speed (COT_opt) was 25.23±4.03 µmol g⁻¹ m⁻¹. A group-wise experiment was conducted with zebrafish (n = 83) swimming at U_opt for 6 h day⁻¹ for 5 days week⁻¹ for 4 weeks vs. zebrafish (n = 84) that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb), insulin-like growth factor 1 receptor a (igf1ra), troponin C (stnnc), slow myosin heavy chain 1 (smyhc1), troponin I2 (tnni2), myosin heavy polypeptide 2 (myhz2) and myostatin (mstnb).

Conclusions/Significance

From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.     

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation

Background

Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models.

Methods

We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1^−/− T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25^hi, CD62L^lo). Additionally, Ceacam1^−/− CD8 T cells had greater expression of the gut-trafficking integrin α₄β₇, though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1^−/− recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1^−/− mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1⁺ lymphoma model was improved in animals receiving Ceacam1^−/− vs. control T cells.

Conclusions

We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Floral thermogenesis of three species of Hydnora (Hydnoraceae) in Africa

Background and Aims

Floral thermogenesis occurs in at least 12 families of ancient seed plants. Some species show very high rates of respiration through the alternative pathway, and some are thermoregulatory, with increasing respiration at decreasing ambient temperature. This study assesses the intensity and regulation of respiration in three species of African Hydnora that represent the Hydnoraceae, an unusual family of holoparasitic plants from arid environments.

Methods

Long-term respirometry (CO₂ production) and thermometry were carried out on intact flowers of H. africana, H. abyssinica and H. esculenta in the field, and short-term measurements were made on floral parts during the protogynous flowering sequence.

Key Results

For H. africana, there was no temperature elevation in either the osmophores or the gynoecial chamber in any phase, and mass-specific respiration rates of the flower parts were low (maximum 8·3 nmol CO₂ g⁻¹ s⁻¹ in osmophore tissue). Respiration tracked ambient and floral temperatures, eliminating the possibility of the inverse relationship expected in thermoregulatory flowers. Hydnora abyssinica flowers had higher respiration (maximum 27·5 nmol g⁻¹ s⁻¹ in the osmophores) and a slight elevation of osmophore temperature (maximum 2·8 °C) in the female stage. Respiration by gynoecial tissue was similar to that of osmophores in both species, but there was no measurable elevation of gynoecial chamber temperature. Gynoecial chamber temperature of H. esculenta could reach 3·8 °C above ambient, but there are no respiration data available. Antheral tissue respiration was maximal in the male phase (4·8 nmol g⁻¹ s⁻¹ in H. africana and 10·3 nmol g⁻¹ s⁻¹ in H. abyssinica), but it did not raise the antheral ring temperature, which showed that thermogenesis is not a by-product of pollen maturation or release.

Conclusions

The exceptionally low thermogenesis in Hydnora appears to be associated with scent production and possibly gynoecial development, but has little direct benefit to beetle pollinators.Key words: Pollination biology, Hydnora, thermogenesis, respiration rate, temperature, flowers, insects     

β-lapachone significantly increases the effect of ionizing radiation to cause mitochondrial apoptosis via JNK activation in cancer cells

Background

β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.

Methodology/Principal Findings

β-lap enhanced the effect of IR to cause clonogenic cells in NQO1⁺-MDA-MB-231 cells but not in NQO1⁻-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1⁺ cells and not in NQO1⁻ cells and it markedly increased IR-induced apoptosis only in NQO1⁺ cells. Combined treatment of NQO1⁺ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.

Conclusions/Significance

β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1⁺ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.     

Vitamin k-antagonists accelerate atherosclerotic calcification and induce a vulnerable plaque phenotype

Background

Vitamin K-antagonists (VKA) are treatment of choice and standard care for patients with venous thrombosis and thromboembolic risk. In experimental animal models as well as humans, VKA have been shown to promote medial elastocalcinosis. As vascular calcification is considered an independent risk factor for plaque instability, we here investigated the effect of VKA on coronary calcification in patients and on calcification of atherosclerotic plaques in the ApoE^−/− model of atherosclerosis.

Methodology/Principal Findings

A total of 266 patients (133 VKA users and 133 gender and Framingham Risk Score matched non-VKA users) underwent 64-slice MDCT to assess the degree of coronary artery disease (CAD). VKA-users developed significantly more calcified coronary plaques as compared to non-VKA users. ApoE^−/− mice (10 weeks) received a Western type diet (WTD) for 12 weeks, after which mice were fed a WTD supplemented with vitamin K₁ (VK₁, 1.5 mg/g) or vitamin K₁ and warfarin (VK₁&W; 1.5 mg/g & 3.0 mg/g) for 1 or 4 weeks, after which mice were sacrificed. Warfarin significantly increased frequency and extent of vascular calcification. Also, plaque calcification comprised microcalcification of the intimal layer. Furthermore, warfarin treatment decreased plaque expression of calcification regulatory protein carboxylated matrix Gla-protein, increased apoptosis and, surprisingly outward plaque remodeling, without affecting overall plaque burden.

Conclusions/Significance

VKA use is associated with coronary artery plaque calcification in patients with suspected CAD and causes changes in plaque morphology with features of plaque vulnerability in ApoE^−/− mice. Our findings underscore the need for alternative anticoagulants that do not interfere with the vitamin K cycle.     

Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis

Background and Aims

Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 µmol m⁻² s⁻¹. However, desiccation at irradiance of 350 µmol m⁻² s⁻¹ induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery.

Methods

Changes in chloroplast structure, CO₂ assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 µmol m⁻² s⁻¹; ML) irradiance were compared with those under low (30 µmol m⁻² s⁻¹; LL) irradiance.

Key Results

Well-watered plants (control) at 100 µmol m⁻² s⁻¹ were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO₂ assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML.

Conclusions

Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.Key words: Haberlea rhodopensis, resurrection plant, electron microscopy, blue–green fluorescence, chlorophyll fluorescence