Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages

The potential use of alpha-cyclodextrin and its hydrophilic alpha-cyclodextrin derivatives (alpha-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production as well as nuclear factor-kappaB (NF-kappaB) activation in macrophages was examined. Of three alpha-CyDs used in the present study, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-alpha production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-alpha-CyD inhibited NF-kappaB translocation into nucleus. These inhibitory effects of DM-alpha-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-alpha-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS.     

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.     

Changes occurring in cortical NO release and brain NO-synthases during a paradoxical sleep deprivation and subsequent recovery in the rat

Variations occurring in cortical nitric oxide (NO) release were analysed with a voltametric method in rats (i) placed in control conditions, (ii) while being paradoxical sleep deprived (PSD), or (iii) recovering from a PSD. Activities of neuronal (nNOS) and inducible (iNOS) NO-synthases as well as nNOS expression were also determined in several brain regions. In baseline conditions, circadian variations in nNOS expression and activity were maximal during the dark period and minimal during the light one for all the structures analysed (frontal cortex, pons and medulla). In the same way, cortical NO release occurred through a circadian rhythm exhibiting maxima and minima during dark and light periods, respectively. In the same experimental conditions, iNOS activity did not exhibit time-dependent changes. The correlative changes observed in baseline conditions between NO release, nNOS expression and activity within the frontal cortex were disrupted during PSD and subsequent recovery. Still again, iNOS activity remained unchanged. Results obtained point out that the tight coupling existing in control conditions between nNOS expression-activity and NO release is disrupted by a PSD and remains affected during the subsequent 24 h recovery. Their significance is discussed.     

A quantitative trait locus for recognition of foreign eggs in the host of a brood parasite

Avian brood parasites reduce the reproductive output of their hosts and thereby select for defence mechanisms such as ejection of parasitic eggs. Such defence mechanisms simultaneously select for counter-defences in brood parasites, causing a coevolutionary arms race. Although coevolutionary models assume that defences and counter-defences are genetically influenced, this has never been demonstrated for brood parasites. Here, we give strong evidence for genetic differences between ejector and nonejectors, which could allow the study of such host defence at the genetic level, as well as studies of maintenance of genetic variation in defences. Briefly, we found that magpies, that are the main host of the great spotted cuckoo in Europe, have alleles of one microsatellite locus (Ase64) that segregate between accepters and rejecters of experimental parasitic eggs. Furthermore, differences in ejection rate among host populations exploited by the brood parasite covaried significantly with the genetic distance for this locus.     

A new inducible adenoviral expression system that responds to inflammatory stimuli in vivo

BACKGROUND: Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. METHODS: A luciferase reporter vector with a hybrid promoter containing the human IL-1beta enhancer region (-3690 to - 2720) and the human CIITA promoter IV (-399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1beta/CIITApIV promoter (Ad-IL1beta/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1beta/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1beta/CIITApIV promoter. RESULTS: The IL1beta/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1beta/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. CONCLUSIONS: Using the IL1beta/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity.     

Optimal investment across different aspects of anti-predator defences

We present a simple model of investment across a suite of different anti-predatory defences. Defences can incur an initial construction cost and and/or may be costly each time they are utilised. Our aim is to use a simple, but general, mathematical model to explore when prey that face a single predatory threat where each attack is of the same nature should invest only in a single defence, and when they should spread their investment across more than one defence. This should help to explain the observed variety of defences that a single prey individual may employ during repeated attacks of a similar nature or even at different stages during one attack. Previous verbal reasoning suggested that prey should specialise in investment in defences that can be utilised early in the predation sequence. Our quantitative model predicts that (depending of the relatively properties of different defences), there may be concentrated investment in early acting, or in late-acting defences, or a spread of investment across both defence types. This variety of predictions is in agreement with the variation in defences shown by natural organisms subjected to repeated predatory attack.     

Effects of wing polyphenism, aphid genotype and host plant chemistry on energy metabolism of the grain aphid, Sitobion avenae

Wing dimorphism has been proposed as a strategy to face trade-offs between flight capability and fecundity. In aphids, individuals with functional wings have slower development and lower fecundity compared with wingless individuals. However, differential maintenance costs between winged and wingless aphids have not been deeply investigated. In the current study, we studied the combined effect of wing dimorphism with the effects of aphid genotypes and of wheat hosts having different levels of chemical defences (hydroxamic acids, Hx) on adult body mass and standard metabolic rates (SMR) of winged and wingless morphs of the grain aphid, Sitobion avenae. We found that wingless aphids had higher body mass than winged aphids and that body mass also increased towards host with high Hx levels. Furthermore, winged aphids showed a plastic SMR in terms of Hx levels, whereas wingless aphids displayed a rigid reaction norm (significant interaction between morph condition and wheat host). These findings suggest that winged aphids have reduced adult size compared to wingless aphids, likely due to costs associated to the development of flight structure in early-life stages. These costs contrast with the absence of detectable metabolic costs related to fuelling and maintenance of the flight apparatus in adults.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.