Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.     

Protection against Chemotaxis in the Anti-Inflammatory Effect of Bioactives from Tomato Ketchup

The consumption of tomato products has been associated with a decreased risk for chronic inflammatory diseases. In this study, the anti-inflammatory potential of tomato ketchup was evaluated by studying the effect of tomato ketchup extracts and bioactives from tomato ketchup on human monocytes and vascular endothelial cells (HUVEC). HUVEC were pre-treated for 1 h with either individual bioactives (7.5 µM lycopene, 1.4 µM α-tocopherol or 55 µM ascorbic acid) or a combination of these three compounds, or with the hydrophilic or lipophilic tomato ketchup extracts or with the two extracts combined. After the pretreatment, the cells were washed and challenged with TNF-α (10 ng/ml) for 6 h. The medium was used for the determination of the release of cytokines and the chemotaxis of monocytes. Inflammatory protein expression and production were assayed with real-time RT-PCR and ELISA. It was found that tomato ketchup extracts significantly reduced gene expression and release of the pro-inflammatory cytokines TNF-α and IL-8 in HUVEC after the inflammatory challenge, whereas the release of the anti-inflammatory cytokine IL-10 was increased. Chemotaxis was effectively impeded as demonstrated by a reduced monocyte migration. This effect correlated with the reduction of IL-8 production in the presence of the test compounds and extracts. The results consistently emphasize the contribution of lycopene to the anti-inflammatory effect of tomato ketchup. Other compounds in tomato ketchup such as α-tocopherol and ascorbic acid appeared to strengthen the anti-inflammatory effect of lycopene. The tomato ketchup extracts subtly interfered with several inflammatory phases that inhibit chemotaxis. Such a pleotropic mode of action exemplifies its potential mitigation of diseases characterized by prolonged low grade inflammation.     

Variation in wood anatomical structure of Douglas-fir defoliated by the western spruce budworm: a case study in the coastal-transitional zone of British Columbia,Canada

Key message

An outbreak of the western spruce budworm temporarily modifies cellular wood anatomy of stem wood in natural and mature Douglas-fir stands impacting wood quality properties.

Abstract

Western spruce budworm (Choristoneura occidentalis Freeman) is a widespread and destructive defoliator of commercially important coniferous forests in western North America. In British Columbia, Canada, Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] is the most important and widely distributed host. Permanent sample plots were established at a number of locations in southern interior at the beginning of a severe western spruce budworm outbreak in the 1970s. Two of the sites were sampled in 2012 to determine whether modifications had occurred in the anatomical characteristics of stem wood formed during outbreak years. We determined that rings formed during the western spruce budworm 1976–1980 outbreak had a significantly lower proportion of latewood, reduced mean cell wall thickness and smaller radial cell diameters. While the cellular characteristics of the earlywood remained fairly constant, significant reductions in lumen area occurred in 1978 and 1979 at each site. Our study shows that western spruce budworm outbreaks not only reduce annual radial growth, but also temporarily modify cellular characteristics in latewood cells, which has implications for wood density and quality in Douglas-fir.     

Solvated docking: introducing water into the modelling of biomolecular complexes

MOTIVATION: Interfacial water, which plays an important role in mediating biomolecular interactions, has been neglected in the modelling of biomolecular complexes. METHODS: We present a solvated docking approach that explicitly accounts for the presence of water in protein-protein complexes. Our solvated docking protocol is based on the concept of the first encounter complex in which a water layer is present in-between the molecules. It mimics the pathway from this initial complex towards the final assembly in which most waters have been expelled from the interface. Docking is performed from solvated biomolecules and waters are removed in a biased Monte Carlo procedure based on water-mediated contact propensities obtained from an analysis of high-resolution crystal structures. RESULTS: We demonstrate the feasibility of this approach for protein-protein complexes representing both 'wet' and 'dry' interfaces. Solvated docking leads to improvements both in quality and scoring. Water molecules are recovered that closely match the ones in the crystal structures. AVAILABILTY: Solvated docking will be made available in the future release of HADDOCK version 2.0 (http://www.nmr.chem.uu.nl/haddock).     

Purinergic receptors involved in the immunomodulatory effects of ATP in human blood

We recently showed that the physiological compound ATP simultaneously inhibited TNF-alpha and stimulated IL-10 release in LPS-PHA stimulated blood. The purpose of the present study was to determine the mechanism involved in the concerted modulatory effect of ATP on TNF-alpha and IL-10. Incubation of blood with ATP in the presence of selective P2 receptor antagonists showed that the stimulatory effect of ATP on IL-10 release was completely annihilated by both 2-MeSAMP (a P2Y12/13 receptor antagonist) and PSB-0413 (a P2Y12 receptor antagonist). On the other hand, the inhibitory effect of ATP on TNF-alpha release was completely reversed by 5'-AMPS (a P2Y11 receptor antagonist) as well as by H-89, an inhibitor of cAMP-activated PKA. The concerted inhibition by ATP of TNF-alpha release via P2Y11 activation and stimulation of IL-10 release via P2Y12 activation implicates a novel approach towards immunomodulation by altering the balance among pro- and anti-inflammatory cytokines.     

Cpt1a silencing in AgRP neurons improves cognitive and physical capacity and promotes healthy aging in male mice

Orexigenic neurons expressing agouti-related protein (AgRP) and neuropeptide Y in the arcuate nucleus (ARC) of the hypothalamus are activated in response to dynamic variations in the metabolic state, including exercise. We previously observed that carnitine palmitoyltransferase 1a (CPT1A), a rate-limiting enzyme of mitochondrial fatty acid oxidation, is a key factor in AgRP neurons, modulating whole-body energy balance and fluid homeostasis. However, the effect of CPT1A in AgRP neurons in aged mice and during exercise has not been explored yet. We have evaluated the physical and cognitive capacity of adult and aged mutant male mice lacking Cpt1a in AgRP neurons (Cpt1a KO). Adult Cpt1a KO male mice exhibited enhanced endurance performance, motor coordination, locomotion, and exploration compared with control mice. No changes were observed in anxiety-related behavior, cognition, and muscle strength. Adult Cpt1a KO mice showed a reduction in gastrocnemius and tibialis anterior muscle mass. The cross-sectional area (CSA) of these muscles were smaller than those of control mice displaying a myofiber remodeling from type II to type I fibers. In aged mice, changes in myofiber remodeling were maintained in Cpt1a KO mice, avoiding loss of physical capacity during aging progression. Additionally, aged Cpt1a KO mice revealed better cognitive skills, reduced inflammation, and oxidative stress in the hypothalamus and hippocampus. In conclusion, CPT1A in AgRP neurons appears to modulate health and protects against aging. Future studies are required to clarify whether CPT1A is a potential antiaging candidate for treating diseases affecting memory and physical activity.     

Nerve-dependent changes in content of ribosomes, polysomes, and nascent peptides in newt limb regenerates.

Regeneration of a newt limb requires a constant supply of adequate amounts of a neuronal contribution at the amputation site. Denervation during the early stages of regeneration precludes its growth and morphogenesis. It has been reported that denervation of a regenerating limb lowers the efficiency of incorporation of radioactive amino acids to 60% of contralateral control levels. To gain more insight into the mechanism responsible for this decrease, we examined the effects of denervation on the size distribution and quantity of regenerate polysomes. We characterized the [³⁵S]methionine-labeled nascent peptidyl-tRNA from polysomes by hydroxyapatite chromatography. Moreover, we show that the labeled nascent peptides on polysomes can serve as a measure to quantitate the relative amounts of ribosomes on polysomes and the relative size of the translational machinery. Thus, we report that [³⁵S]methionine-labeled nascent polypeptides on polysomes from denervated regenerates contain about 48% less radioactivity than those from controls. Despite decreased incorporation of [³⁵S]methionine into nascent peptides, the relative distribution of radioactivity across linear sucrose gradients is not significantly altered by denervation. Studies of polysomes labeled with [³H]uridine prior to denervation indicate that ribosome content is depressed by denervation. Our results suggest that the nerve-dependent decrease in protein synthesis is mediated by decreasing the number of ribosomes active in protein synthesis. In addition, similarities in the ratios of free monosomes to polysomes and the relative size distribution of polypeptides between denervated and innervated regenerates indicate that in denervated regenerates the number of translatable mRNA molecules decreases in a coordinate manner with the number of ribosomes active in protein synthesis.     

Identification of structural characteristics of some potential H2-receptor antagonists that determine the interaction with rat hepatic P-450

Several potential H2-receptor antagonists have been tested in vitro, using liver microsomal preparations from untreated rats, in order to study their interaction with P-450. The aim of this investigation was to establish structure-activity relationships for the P-450-inhibition developed by cimetidine and related drugs. Most of the compounds tested demonstrate an inhibitory activity and a binding ability to P-450, via type II (ligand type) binding. Our results strongly indicate that the cyano-guanidine moiety is an essential structural feature for both the inhibition of a ferrocytochrome P-450-metabolic intermediate complex formation occurring during the metabolism of tofenacine, and the binding of the compounds to the heme iron of P-450. The presence of an imidazole group is not necessary for these activities. Furthermore, it is pointed out that the lipophilic character of the cyano-guanidine side chain contributes to the interaction of the test compounds with P-450, since a trend for a parabolic relationship between lipophilicity and inhibitory activity or binding ability is observed. Finally, under the experimental conditions used, no increase of the inhibitory activity of cimetidine on the metabolism of tofenacine and 7-ethylresorufin is observed after preincubation of rat liver microsomes with cimetidine, confirming earlier results in similar studies.