20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system and kidney. They modulate ion transport and gene expression, producing vasorelaxation as well as anti-inflammatory and pro-fibrinolytic effects. EETs are incorporated into the sn-2 position of phospholipids and are rapidly mobilized when a cell is treated with a Ca(2+) ionophore, suggesting that they may play a role in phospholipid-mediated signal transduction processes. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs), and inhibition of sEH is a potential approach for enhancing the biological activity of EETs. EETs also undergo chain-elongation and beta-oxidation, and the accumulation of partial beta-oxidation products increases when sEH is inhibited. Some functional effects of EETs occur through activation of either the guanine nucleotide binding protein Galphas or the Src signal transduction pathways, suggesting that EETs act by binding to membrane receptors. However, other evidence indicates that the modulation of gene expression occurs through an intracellular action of EETs. Because of the diversity of biochemical and functional responses produced by EETs, it is doubtful that a single mechanism or signal transduction pathway can account for all of their actions.     

Fas and Fas ligand mutations inhibit autoantibody production in pristane-induced lupus

Mutations of Fas (lpr) or Fas ligand (gld) cause a limited lupus-like syndrome in B6 mice by interfering with the deletion of autoreactive B and/or T cells. A more generalized lupus syndrome reminiscent of that of MRL mice can be induced in nonautoimmune strains by pristane, which causes a nonspecific inflammatory response in the peritoneal cavity. We hypothesized that, as in MRL mice, the lpr and gld mutations might accelerate lupus in pristane-treated mice. Pristane-treated B6 mice developed anti-nRNP/Sm, Su, and ribosomal P Abs, but little anti-ssDNA or chromatin. In contrast, B6/lpr and B6/gld mice spontaneously developed anti-ssDNA/chromatin Abs, but not anti-nRNP/Sm/Su/ribosomal P. Unexpectedly, B6/lpr and B6/gld mice were highly resistant to the induction by pristane of IgM anti-ssDNA (2 wk) and IgG anti-nRNP/Sm/Su/ribosomal P autoantibodies (6 mo), suggesting that intact Fas signaling is necessary. Interestingly, pristane did not enhance IgG chromatin Ab production in B6/lpr or B6/gld mice, suggesting that it did not influence the production of autoantibodies that develop spontaneously in the setting of Fas deficiency. Pristane treatment also decreased lymphoproliferation in B6/lpr mice. Increased production of IL-12 was associated consistently with the production of anti-nRNP/Sm/Su/ribosomal P as well as anti-DNA/chromatin. In contrast, production of anti-DNA/chromatin Abs was associated with IL-6 overproduction in pristane-treated mice, but not in lpr mice. The data strongly support the idea that different subsets of autoantibodies are regulated differentially by cytokine stimulation and/or Fas signaling.     

Direct measurement of the lumped constant for 2-deoxy-[1-(14)C]glucose in vivo in human skeletal muscle

The lumped constant (LC) is used to convert the clearance rate of 2-deoxy-D-glucose (2-DG(CR)) to that of glucose (Glc(CR)). There are currently no data to validate the widely used assumption of an LC of 1.0 for human skeletal muscle. We determined the LC for 2-deoxy-[1-(14)C]glucose (2-DG) in 18 normal male subjects (age, 29+/- 2 yr; body mass index, 24.8+/-0.8 kg/m(2)) after an overnight fast and during physiological (1 mU x kg(-1) x min(-1) insulin infusion for 180 min) and supraphysiological (5 mU x kg(-1) x min(-1) insulin infusion for 180 min) hyperinsulinemic conditions. Normoglycemia was maintained with the euglycemic clamp technique. The LC was measured directly with the use of a novel triple tracer-based method. [3-(3)H]glucose, 2-[1-(14)C]DG, and [(12)C]mannitol (Man) were injected as a bolus into the brachial artery. The concentrations of [3-(3)H]glucose and 2-[1-(14)C]DG (dpm/ml plasma) and of Man (micromol/l) were determined in 50 blood samples withdrawn from the ipsilateral deep forearm vein over 15 min after the bolus injection. The LC was calculated by a formula involving blood flow calculated from Man and the Glc(CR) and 2-DG(CR). The LC averaged 1.26+/-0.08 (range 1.06-1.43), 1.15+/-0.05 (0.99-1.39), and 1.18+/-0.05 (0.97-1.37) under fasting conditions and during the 1 and 5 mU x kg(-1). min(-1) insulin infusions (not significant between the different insulin concentrations, mean LC = 1.2, P<0.01 vs. 1.0). We conclude that, in normal subjects, the LC for 2-DG in human skeletal muscle is constant over a wide range of insulin concentrations and averages 1. 2.     

Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.     

Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-x_L is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-x_L, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na⁺/H⁺ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-x_L deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-x_L deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-x_L is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-x_L deamidation. The conservation of such histidines implies that human Bcl-x_L is in essence “designed” to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-x_L.     

Factors affecting the biological control of the spiralling whitefly Aleurodicus dispersus (Hemiptera: Aleyrodidae) in banana plantations in Costa Rica: a study case

ABSTRACT

A study was conducted to reveal the causes for Aleurodicus dispersus outbreak in commercial banana plantations in Costa Rica. Biotic and abiotic factors affecting the abundance of the spiralling whitefly were determined. Four nymph parasitoids and nine predators were found to affect A. dispersus population. Seventeen families of host plants for A. dispersus in the banana area were identified, allowing the whitefly to reach a high reproductive and dispersal rate and pest status in short periods. The use of nematicides during the dry season was the key factor for mortality of natural enemies responsible for maintaining pest populations under economic levels.     

Modest Gaseous Nitrogen Losses Point to Conservative Nitrogen Cycling in a Lowland Tropical Forest Watershed

Primary tropical rainforests are generally considered to be relatively nitrogen (N) rich, with characteristically large hydrologic and gaseous losses of inorganic N. However, emerging evidence suggests that some tropical ecosystems can exhibit tight N cycling, with low biologically available losses. In this study, we combined isotopic data with a well-characterized watershed N mass balance to close the N budget and characterize gaseous N losses at the ecosystem scale in a lowland tropical rainforest on the Osa Peninsula in southwestern Costa Rica. We measured δ¹⁵N and δ¹⁸O of nitrate (NO₃ ^?) in precipitation, surface, shallow and deep soil lysimeters and stream water biweekly for 1 year. Enrichment of both isotopes indicates that denitrification occurs predominantly as NO₃ ^? moves from surface soil down to 15 cm depth or laterally to stream water, with little further processing in deeper soil. Two different isotopic modeling approaches suggested that the gaseous fraction comprises 14 or 32% of total N loss (2.7 or 7.5 kg N ha^?1 y^?1), though estimates are sensitive to selection of isotopic fractionation values. Gas loss estimates using the mass balance approach (3.2 kg N ha^?1 y^?1) fall within this range and include N₂O losses of 0.9 kg N ha^?1 y^?1. Overall, gaseous and soluble hydrologic N losses comprise a modest proportion (~ 25%) of the total N inputs to this ecosystem. By contrast, relatively large, episodic hydrologic losses of non-biologically available particulate N balance the majority of N inputs and may contribute to maintaining conservative N cycling in this lowland tropical forest. Similar patterns of N cycling may occur in other tropical forests with similar state factor combinations—high rainfall, steep topography, relatively fertile soils—such as the western arc of the Amazon Basin and much of IndoMalaysia, but this hypothesis remains untested.     

Down‐regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H₂O₂ in the 10 μm range. This concentration range seems to be necessary to activate H₂O₂‐dependent signalling pathways stimulating the expression of H₂O₂ responsive genes, such as the heat shock protein HSP22C. Catalase knock‐down mutants had lost the transient accumulation of H₂O₂, suggesting that a decrease in catalase activity was the key element for establishing a transient H₂O₂ burst. Catalase was inactivated by a one‐electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H₂O₂, inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast‐directed protection enzymes.     

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation

Background

The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males.

Results

Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity.

Conclusions

This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-799) contains supplementary material, which is available to authorized users.