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Zymosan, an immunogenic cell wall extract of Saccharomyces cerevisiae has potential for use as an experimental pyrogen. However, the short-lived sickness responses noted with intraperitoneal and intra-articular administration of zymosan limits investigations on the long-term effectiveness of antipyretic drugs. Thus, there remains a need to establish an alternative route of zymosan administration that could induce long-lived fevers and inflammation. We injected male Sprague Dawley rats (250–300 g) subcutaneously with zymosan (30 or 300 mg/kg) or saline; n = 7–8. We measured core body temperature, cage activity, food intake and body mass for 24 h after injection. Blood and brain samples were collected at 2, 8, and 18 h after injection. Zymosan (300 mg/kg) induced fever, lethargy, and anorexia, which lasted for 24 h. Zymosan-induced sickness responses were accompanied by increased blood plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α; activation of inflammatory transcription factors (nuclear factor (NF) for IL-6, signal transducer and activator of transcription (STAT)-3, and NF-κB) in the hypothalamus and circumventricular organs; and increased hypothalamic mRNA expression of TNF-α, IL-1β, and IL-6 and rate-limiting enzymes for prostaglandin synthesis. Our results confirm the suitability of subcutaneous administration of zymosan for screening antipyretic and anti-inflammatory drugs in rats.

GnRH receptor mutations, Glu2.53(90)Lys and Glu2.53(90)Asp, cause congenital hypogonadotropic hypogonadism. The Glu2.53(90) side-chain has been proposed to form an intramolecular salt-bridge with Lys3.32(121), but conserved intramolecular interaction networks in G protein-coupled receptor crystal structures predict that it interacts with Ser3.35(124) and Trp6.48(280). We investigated interhelical interactions of Glu2.53(90) that stabilise GnRH receptor folding using functional analyses and computational modelling of mutant receptors. The Glu2.53(90)Asp mutant was non-functional, but mutants with hydrophobic amino acids or Arg substituted for Glu2.53(90) were functional, excluding a salt-bridge interaction. The Glu2.53(90)Arg and Trp6.48(280)Arg mutants had decreased affinity for GnRH. Models showed that congenital Glu2.53(90)Lys and Glu2.53(90)Asp mutations disrupt interactions with Ser3.35(124) and Trp6.48(280) respectively, whereas the Glu2.53(90)Arg and Trp6.48(280)Arg mutations preserve intramolecular contacts, but increase distance between the transmembrane helices. Our results show that disruption of interhelical contacts that are conserved in G protein-coupled receptors accounts for the effects of some disease-associated GnRH receptor mutations.

Brewer’s yeast, derived from the yeast species Saccharomyces cerevisiae (S. cerevisiae), is commonly used for inducing pyrexia in pharmacological studies screening antipyretics in rats. Despite its widespread use, the peripheral and central inflammatory response associated with Brewer’s yeast-induced fever and sickness behavior in rats has not been investigated. Thus, we injected male Sprague-Dawley rats (150–200 g) subcutaneously with a high (4 g/kg, n = 9), medium (2 g/kg, n = 5) or low (0.4 g/kg, n = 6) dose of Brewer’s yeast solution or saline (0.9%, n = 6) and measured core body temperature, cage activity, food intake and body mass for six days after injection. Blood and brain samples were collected at 2, 8, 18 and 72 h after injection; n = 5–7 per time point. Brewer’s yeast administration dose-dependently induced fever, lethargy, anorexia and body mass stunting that was accompanied by increased blood plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and activation of inflammatory transcription factors (nuclear factor (NF) for interleukin-6, signal transducer and activator of transcription (STAT)-3, and NF-κB)) in the hypothalamus and circumventricular organs. The increased activation of transcription factors following Brewer’s yeast administration was accompanied by increased hypothalamic mRNA expression of TNF-α, IL-1β and IL-6 and rate-limiting enzymes for prostaglandin synthesis. Our results show that subcutaneous administration of S. cerevisae induces prolonged fever, anorexia and lethargy that is accompanied by a pronounced increase in the synthesis of pro-inflammatory cytokines, key prostaglandin synthesizing enzymes and transcription factors, in the periphery and brain.

Stress-induced hyperthermia following rectal thermometry is reported in normothermic rats, but appears to be muted or even absent in febrile rats. We therefore investigated whether the use of rectal thermometry affects the accuracy of temperature responses recorded in normothermic and febrile rats. Using intra-abdominally implanted temperature-sensitive radiotelemeters we measured the temperature response to rectal temperature measurement in male Sprague Dawley rats (~ 200 g) injected subcutaneously with Brewer's yeast (20 ml/kg of a 20% Brewer's yeast solution = 4000 mg/kg) or saline (20 ml/kg of 0.9% saline). Rats had been pre-exposed to, or were naive to rectal temperature measurement before the injection. The first rectal temperature measurement was taken in the plateau phase of the fever (18 h after injection) and at hourly intervals thereafter. In normothermic rats, rectal temperature measurement was associated with an increase in abdominal temperature (0.66 ± 0.27 °C) that had a rapid onset (5–10 min), peaked at 15–20 min and lasted for 35–50 min. The hyperthermic response to rectal temperature measurement was absent in febrile rats. Exposure to rectal temperature measurement on two previous occasions did not reduce the hyperthermia. There was a significant positive linear association between temperatures recorded using the two methods, but the agreement interval identified that rectal temperature measured with a thermocouple probe could either be 0.7 °C greater or 0.5 °C lower than abdominal temperature measured with radiotelemeter. Thus, due to stress-induced hyperthermia, rectal thermometry does not ensure accurate recording of body temperature in short-spaced, intermittent intervals in normothermic and febrile rats.

Objective The current study aimed to investigate the impact of high-fat diets composed of different animal and vegetable fat sources on serum metabolic health markers in Japanese quail, as well as the overall lipid content and fatty acid profiles of the edible bird tissues following significantly increased dietary lipid supplementation. Methods Fifty seven male quail were divided into six groups and fed either a standard diet or a diet enriched with one of five different fats (22% coconut oil, lard, palm oil, soybean oil, or sunflower oil) for 12 weeks. The birds were subjected to an oral glucose tolerance test following the feeding period, after which they were euthanized and blood, liver, breast, and thigh muscle samples collected. Total fat content and fatty acid profiles of the tissue samples, as well as serum uric acid, triglyceride, cholesterol, total protein, albumin, aspartate transaminase, and total bilirubin concentrations were assessed. Results High-fat diet feeding had no significant effects on the glucose tolerance of the birds. Dietary fatty acid profiles of the added fats were reflected in the lipid profiles of both the liver and breast and thigh muscle tissues, indicating successful transfer of dietary fatty acids to the edible bird tissues. The significantly increased level of lipid inclusion in the diets of the quail used in the present study was unsuccessful in increasing the overall lipid content of the edible bird tissues. Serum metabolic health markers in birds on the high-fat diets were not significantly different from those observed in birds on the standard diet. Conclusion Thus, despite the various high-fat diets modifying the fatty acid profile of the birds' tissues, unlike in most mammals, the birds maintained a normal health status following consumption of the various high-fat diets.

We investigated the effect of oral curcumin, on bone health of rats fed a high-fructose diet. Suckling pups (males = 65, females = 63) were gavage with 0.5% DMSO, curcumin (500 mg/kg), fructose (20%, w/v) or a combination of curcumin and fructose daily from postnatal days 6 to 21. Then the rats were weaned onto normal rat feed for six weeks and each group was sub-divided into two subgroups: one had plain tap water and the other had fructose (20%, w/v) to drink. Blood was assayed for plasma total osteocalcin. Morphometry and radiographic bone density assessments were made on the femora and tibiae. The lengths, masses and Seedor indices of the bones were similar (p > 0.05, ANOVA) across the groups. Males that received curcumin with or without fructose during suckling and weaned onto a high-fructose diet had lower (p ≤ 0.05, ANOVA) osteocalcin concentration versus the other males. Similarly, in females rats, curcumin alone or administered with fructose resulted in lower (p ≤ 0.05, ANOVA) osteocalcin concentration versus female rats administered the vehicle control. Neonatal curcumin-induced decrease in plasma total osteocalcin concentration may predispose to adverse consequences on glucose metabolism and bone health.

GnRH receptor mutations, Glu

The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization.

There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells. We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system.Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using (1)H and (13)C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTT assay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug.Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca(2+) dependent uptake of nanoparticles by the macrophages.PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy.

1. Synaptotagmin has been reported to function in clathrin-mediated endocytosis. Here, we investigated its involvement in agonist-stimulated internalization of M4 muscarinic acetylcholine receptors exogenously expressed in human embryonic kidney (HEK-293 tsA201) cells. 2. Synaptotagmin I was present at low levels in these cells, and when overexpressed resided at the plasma membrane. 3. Synaptotagmin overexpression alone did not affect receptor internalization, but 'rescued' internalization that had been inhibited by either dominant-negative dynamin-1 or dominant-negative arrestin-2. Both normal and 'rescued' internalization were sensitive to inhibitors of clathrin-mediated endocytosis, but not to inhibitors of the function of caveolae. 4. There was no increase in AP-2 recruitment to the plasma membrane in cells overexpressing synaptotagmin. However, a mutant form of the receptor lacking a potential AP-2 recruitment motif, while being internalized normally in response to agonist stimulation, was not rescued by synaptotagmin in cells expressing dominant-negative dynamin or arrestin. 5. A mutant form of synaptotagmin (K326,327A), which binds phosphatidylinositol-4,5-bisphosphate (PIP2) much more weakly than the wild-type protein, did not rescue internalization. Furthermore, internalization was inhibited by the PH domain of phospholipase C-delta1, which sequesters PIP2, and synaptotagmin was now unable to rescue. 6. We propose that AP-2 binding to the C-terminal tail of the receptor is not normally required for its endocytosis, but that the synaptotagmin-mediated rescue involves the formation of a ternary complex with the receptor and AP-2. PIP2 might play a role as an intermediary in the formation of this complex.

Summary Poultry diets are formulated with additional animal fat or vegetable oils to improve growth rate and feed conversion efficiency. High-fat diet feeding in rats and fish has been shown to result in alterations in the phospholipid composition and cholesterol content of the erythrocyte membrane, in turn affecting erythrocyte osmotic fragility. In contrast, the few studies performed using high-fat diet feeding in avian species show no changes in erythrocyte osmotic fragility. This study made use of the Japanese quail as no data exists on investigation of this species with respect to high-fat diet feeding and erythrocyte osmotic fragility. Fifty-seven male quail were randomly divided into six groups and fed either a standard diet (commercial poultry feed) or one of five high-fat diets (commercial poultry feed with 22% of either coconut oil, lard, palm oil, soya bean oil or sunflower oil on a weight/weight basis) for 12 weeks. All birds on the high-fat diets were significantly heavier (p

The muscarinic M4 receptor for acetylcholine was tagged at its C terminus with green fluorescent protein (GFP) and expressed in NG108-15 cells, which normally express this receptor subtype. The binding affinity of the antagonist N-methylscopolamine was not significantly affected by the presence of the GFP tag, whereas the affinity of the receptor for the agonist carbachol was reduced by four-fold. Stimulation of the tagged receptor resulted in inhibition of adenylyl cyclase. Following agonist stimulation, the tagged receptor was slowly internalized, and became partially co-localized with the endosomal marker Texas Red-transferrin after 30 min. There was little co-localization with the lysosomal marker 1gp120 even after 60 min of internalization. Finally, the tagged receptor, unlike the endogenous receptor, failed to recycle to the plasma membrane on removal of the agonist. We conclude that the GFP-tagged muscarinic M4 receptor does not traffic normally in NG108-15 cells, most likely because of its gross overexpression.

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.

Summary To investigate the progressive effects of a high-fat diet on erythrocyte osmotic fragility, growth performance and serum lipid concentrations in Guinea fowl and Muscovy ducks, 36 Guinea fowl and 36 Muscovy ducks were divided into two groups, for each species, and fed either a standard (STD = commercial poultry feed) or high-fat diet (HFD = commercial poultry feed with 20% palm oil and 2% lard) for up to 12 weeks. After 4, 8 and 12 weeks on the diets, six birds from each group were euthanized and blood samples collected. Osmotic fragility was assessed by measuring the haemoglobin released by erythrocytes placed in serially diluted solutions of phosphate-buffered saline, spectrophotometrically. Serum triglyceride and cholesterol concentrations were also determined. Fragiligrams from erythrocytes from both species of birds on the HFD were not different to those on the STD. However, Muscovy duck erythrocytes were more resistant to haemolysis compared with Guinea fowl erythrocytes. Final body mass and serum triglyceride levels were not significantly different (p > 0.05, anova) between the birds in the HFD and STD groups, for both species of birds. In contrast, serum cholesterol levels were significantly higher in birds on the HFD compared with those on the STD, after 4, 8 and 12 weeks of feeding, for both species of birds. Feeding Guinea fowl and Muscovy ducks a high-fat diet for up to 12 weeks resulted in hypercholesterolaemia but had no effect on final body mass, erythrocyte osmotic fragility or serum triglyceride concentrations in either bird species.

The V2 vasopressin receptor, a member of the rhodopsin subfamily of GPCRs, mediates arginine vasopressin control of water reabsorption in the kidney by activating Gs. Requirement of the third intracellular loop of the V2R for Gs activation was identified by introducing V2R segments into the Gq coupled V1aR [Liu, J. and Wess, J. (1996) J. Biol. Chem. 271, 8772–8778]; the same approach recognized glutamate 231 and glutamine 225 at the amino terminus of loop 3i as being needed for signal transduction. Site-directed mutagenesis of the V2R confirmed their observations. Recently, we found that a positively charged amino acid at codon 268 is essential for V2R expression, although a double-mutant bearing lysine at position 231 and glutamic acid at position 268 was expressed at higher levels than the wild type V2R and displayed unchanged ligand-binding affinity. Ligand-induced internalization and phosphorylation of the double-mutant receptor was indistinguishable from that observed with the wild type protein but signaling activity was greatly diminished. The data suggested these two amino acids might interact with each other and might play a role in promoting GDP/GTP exchange.

1. Synaptotagmin has been reported to function in clathrin-mediated endocytosis. Here, we investigated its involvement in agonist-stimulated internalization of M4 muscarinic acetylcholine receptors exogenously expressed in human embryonic kidney (HEK-293 tsA201) cells. 2. Synaptotagmin I was present at low levels in these cells, and when overexpressed resided at the plasma membrane. 3. Synaptotagmin overexpression alone did not affect receptor internalization, but 'rescued' internalization that had been inhibited by either dominant-negative dynamin-1 or dominant-negative arrestin-2. Both normal and 'rescued' internalization were sensitive to inhibitors of clathrin-mediated endocytosis, but not to inhibitors of the function of caveolae. 4. There was no increase in AP-2 recruitment to the plasma membrane in cells overexpressing synaptotagmin. However, a mutant form of the receptor lacking a potential AP-2 recruitment motif, while being internalized normally in response to agonist stimulation, was not rescued by synaptotagmin in cells expressing dominant-negative dynamin or arrestin. 5. A mutant form of synaptotagmin (K326,327A), which binds phosphatidylinositol-4,5-bisphosphate (PIP2) much more weakly than the wild-type protein, did not rescue internalization. Furthermore, internalization was inhibited by the PH domain of phospholipase C-delta1, which sequesters PIP2, and synaptotagmin was now unable to rescue. 6. We propose that AP-2 binding to the C-terminal tail of the receptor is not normally required for its endocytosis, but that the synaptotagmin-mediated rescue involves the formation of a ternary complex with the receptor and AP-2. PIP2 might play a role as an intermediary in the formation of this complex.