Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Conservation of transition state structure in fast folding peripheral subunit-binding domains

Φ-Value analysis was used to characterise the structure of the transition state (TS) for folding of POB L146A Y166W, a peripheral subunit-binding domain that folds in microseconds. Helix 2 was structured in the TS with consolidating interactions from the structured loop that connects the two α-helices. This distribution of Φ-values was very similar to that determined for E3BD F166W, a homologue with high sequence and structural similarity. The extrapolated folding rate constants in water at 298 K were 210,000 s^− 1 for POB and 27,500 s^− 1 for E3BD. A contribution to the faster folding of POB came from its having significantly greater helical propensity in helix 2, the folding nucleus. The folding rate also appeared to be influenced by differences in the sequence and structural properties of the loop connecting the two helices. Unimodal downhill folding has been proposed as a conserved, biologically important property of peripheral subunit-binding domains. POB folds five times faster and E3BD folds slower than a proposed limit of 40,000 s^− 1 for barrier-limited folding. However, experimental evidence strongly suggests that both POB L146A Y166W and E3BD F166W fold in a barrier-limited process through a very similar TS ensemble.     

The blood-brain barrier significantly limits eflornithine entry into Trypanosoma brucei brucei infected mouse brain

Drugs to treat African trypanosomiasis are toxic, expensive and subject to parasite resistance. New drugs are urgently being sought. Although the existing drug, eflornithine, is assumed to reach the brain in high concentrations, little is known about how it crosses the healthy and infected blood-brain barrier. This information is essential for the design of drug combinations and new drugs. This study used novel combinations of animal models to address these omissions. Eflornithine crossed the healthy blood-CNS interfaces poorly, but this could be improved by co-administering suramin, but not nifurtimox, pentamidine or melarsoprol. Work using a murine model of sleeping sickness demonstrated that Trypanosoma brucei brucei crossed the blood-CNS interfaces, which remained functional, early in the course of infection. Concentrations of brain parasites increased during the infection and this resulted in detectable blood-brain barrier, but not choroid plexus, dysfunction at day 28 post-infection with resultant increases in eflornithine brain delivery. Barrier integrity was never restored and the animals died at day 37.9 +/- 1.2. This study indicates why an intensive treatment regimen of eflornithine is required (poor blood-brain barrier penetration) and suggests a possible remedy (combining eflornithine with suramin). The blood-brain barrier retains functionality until a late, possibly terminal stage, of trypanosoma infection.     

High temporal resolution of amino acid levels in rat nucleus accumbens during operant ethanol self-administration: involvement of elevated glycine in anticipation

Capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF) provides 15-s temporal resolution of amino acid levels in microdialysate, which, for the first time, allows almost real time measurement of changes during episodes of behavior. We trained Sprague-Dawley rats to self-administer either 10% ethanol-containing gelatin or non-alcoholic gelatin in a typical operant chamber. After rats reached stable daily levels of responding, microdialysis probes were inserted into nucleus accumbens and samples were collected before, during and after operant sessions with on-line analysis via CE-LIF. During the first 15 min of the operant session, there was a significant increase in taurine that correlated with the amount of ethanol consumed ( R ² = 0.81) but no change in rats responding for plain gel. There were large, consistent increases in glycine in both the ethanol and plain gel groups which correlated with the amount of gel consumed. A smaller increase was observed in rats with free non-operant access to plain gel compared to the increase seen with the same amount of gel consumed under operant conditions. When rats were given a time out after each delivery of gel in the operant protocol, the greatest increase of glycine was obtained with the longest time out period. Thus, increases in glycine in nucleus accumbens appear to be related to anticipation of reinforcement.     

PHOTOSYNTHESIS AND PRODUCTION OF HYDROGEN PEROXIDE BY SYMBIODINIUM (PYRRHOPHYTA) PHYLOTYPES WITH DIFFERENT THERMAL TOLERANCES1

Occurrences whereby cnidaria lose their symbiotic dinoflagellate microalgae (Symbiodinium spp.) are increasing in frequency and intensity. These so‐called bleaching events are most often related to an increase in water temperature, which is thought to limit certain Symbiodinium phylotypes from effectively dissipating absorbed excitation energy that is otherwise used for photochemistry. Here, we examined photosynthetic characteristics and hydrogen peroxide (H₂O₂) production, a possible signal involved in bleaching, from two Symbiodinium types (a thermally “tolerant” A1 and “sensitive” B1) representative of cnidaria–Symbiodinium symbioses of reef‐building Caribbean corals. Under steady‐state growth at 26°C, a higher efficiency of PSII photochemistry, rate of electron turnover, and rate of O₂ production were observed for type A1 than for B1. The two types responded very differently to a period of elevated temperature (32°C): type A1 increased light‐driven O₂ consumption but not the amount of H₂O₂ produced; in contrast, type B1 increased the amount of H₂O₂ produced without an increase in light‐driven O₂ consumption. Therefore, our results are consistent with previous suggestions that the thermal tolerance of Symbiodinium is related to adaptive constraints associated with photosynthesis and that sensitive phylotypes are more prone to H₂O₂ production. Understanding these adaptive differences in the genus Symbiodinium will be crucial if we are to interpret the response of symbiotic associations, including reef‐building corals, to environmental change.     

Genetic, biochemical, and physiological characterization of spinosad resistance in Plutella xylostella (Lepidoptera: Plutellidae)

Bioassays (at generation G2) with a newly collected field population (designated MN) of Plutella xylostella (L.) (Lepidoptera: Plutellidae) from Multan, Pakistan, indicated resistance to spinosad, indoxacarb, deltamethrin, abamectin, and acetamiprid. At G2 the field-derived population was divided into two subpopulations, one was selected (G2 to G11) with spinosad (Spino-SEL), whereas the second was left unselected (UNSEL). A significant reduction in the resistance ratio for each compound was observed in UNSEL at G12, indicating that the observed resistance to each insecticide was unstable. For Spino-SEL, bioassays at G12 found that selection with spinosad gave a resistance ratio of 283 compared with MN at G2. The resistance to indoxacarb and acetamiprid in the Spino-SEL population increased to 13- and 67-fold, respectively, compared with MN at G2. The toxicity of deltamethrin to Spino-SEL was similar to its toxicity to the MN population at G2. This suggests that spinosad selection maintained the otherwise unstable resistance to the compound. In contrast, resistance to abamectin decreased significantly from G2 to G12 in Spino-SEL. Logit regression analysis of F1 reciprocal crosses between Spino-SEL and the susceptible Lab-UK indicated that resistance to spinosad was inherited as an autosomal, incompletely recessive trait. The spinosad resistance allele significantly delays the developmental time, reduced pupal weight, number of eggs laid, and number of eggs hatched compared with Lab-UK. Further analysis suggests Spino-SEL exhibited a significantly lower intrinsic rate of population increase (r(m)) to all other populations tested.     

Regulation of alternative macrophage activation by galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.     

Fibroblast growth factor receptor 2 phosphorylation on serine 779 couples to 14-3-3 and regulates cell survival and proliferation

The fibroblast growth factors (FGFs) exert their diverse (or pleiotropic) biological responses through the binding and activation of specific cell surface receptors (FGFRs). While FGFRs are known to initiate intracellular signaling through receptor tyrosine phosphorylation, the precise mechanisms by which the FGFRs regulate pleiotropic biological responses remain unclear. We now identify a new mechanism by which FGFR2 is able to regulate intracellular signaling and cellular responses. We show that FGFR2 is phosphorylated on serine 779 (S779) in response to FGF2. S779, which lies adjacent to the phospholipase Cgamma binding site at Y766, provides a docking site for the 14-3-3 phosphoserine-binding proteins and is essential for the full activation of the phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase pathways. Furthermore, S779 signaling is essential for promoting cell survival and proliferation in both Ba/F3 cells and BALB/c 3T3 fibroblasts. This new mode of FGFR2 phosphoserine signaling via the 14-3-3 proteins may provide an increased repertoire of signaling outputs to allow the regulation of pleiotropic biological responses. In this regard, we have identified conserved putative phosphotyrosine/phosphoserine motifs in the cytoplasmic domains of diverse cell surface receptors, suggesting that they may perform important functional roles beyond the FGFRs.