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In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.

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Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga, Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation--16:5, 18:4, 20:5, 20:6, and 22:7--was accomplished by organic extraction followed by a combination of vacuum and high-performance liquid chromatography. One of these that was a novel substance (22:7) was characterized as 4Z,7Z,9E,11E,13Z,16Z,19Z-do cosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoi c acid) resulted in substantially increased synthesis of unique tetraene compounds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.

End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.

The neurohormone arginine vasotocin (AVT) in non mammalian vertebrates is homologous to arginine vasopressin (AVP) in mammals. Its actions are mediated via G protein-coupled receptors that belong to the vasotocin/mesotocin family. Because of the known regulatory effects of nonapeptide hormones on anterior pituitary functions, receptor subtypes in that family have been proposed to be located in anterior pituitary cells. Recently, an avian vasotocin receptor subtype designated VT4R has been cloned, which shares 69% sequence homology with a human vasopressin receptor, the V1aR. In the present study, a polyclonal antibody to the VT4R was developed and validated to confirm its specificity to the VT4R. The antibody was used to test the hypothesis that the VT4R is present in the avian anterior pituitary and is specifically associated with certain cell types, where its expression is modulated by acute stress. Western blotting of membrane protein extracts from pituitary tissue, the use of HeLa cells transfected with the VT4R and peptide competition assays all confirmed the specificity of the antibody to the VT4R. Dual-labelling immunofluorescence microscopy was utilised to identify pituitary cell types that contained immunoreactive VT4R. The receptor was found to be widely distributed throughout the cephalic lobe but not in the caudal lobe of the anterior pituitary. Immunoreactive VT4R was associated with corticotrophs. Approximately 89% of immunolabelled corticotrophs were shown to contain the VT4R. The immunoreactive VT4R was not found in gonadotrophs, somatotrophs or lactotrophs. To determine a possible functional role of the VT4R and previously characterised VT2R, gene expression levels in the anterior pituitary were determined after acute immobilisation stress by quantitative reverse transcriptase-polymerase chain reaction. The results showed a significant increase in plasma corticosterone levels (three- to four-fold), a significant reduction of VT4R mRNA and an increase of VT2R mRNA (P < 0.05) in acutely immobilised chicks compared to controls. The data suggest a role of the VT4R in the avian stress response.

Fish species exhibit great diversity rating of aging (from negligible to rapid), which gives a unique possibility for the discovery of the molecular mechanisms that determine the differences in the rate of aging. A mass spectrometric metabolic profiling of skeletal muscle of fish with various aging rates was carried out by direct injection to a quadrupole time-of-flight mass spectrometer. The first group includes long-lived fish species (pike (Esox Lucius) and sterlet (Acipenser ruthenus); the second group—species with gradual senescence such as that observed in many mammalian species of similar size (zander (Sandra lucioperca) and perch (Perca fluviatilis)) and the third group—species with very short life cycle (chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha)). Multivariate analysis of metabolic profiles allowed the detecting of about 80 group-specific features associated with amino acids, lipids, biogenic amines, intermediates of glycolysis, glycogenolysis, and citric acid cycle. Possible roles in the aging process are hypothesized for the biochemical pathways of the metabolites that were altered in the different groups.

There are a number of different animals that belong to long- and short-lived species and show a various rate of ageing, providing an ideal model to investigate mechanisms of longevity. In this work, a metabolome profiling of blood plasma from fishes with various ageing rates—negligible (Pike Esox Lucius and Sterlet Acipenser ruthenus), gradual (Zander Sander lucioperca and Perch Perca fluviatilis) and rapid (Chum Salmon Oncorhynchus keta and Pink Salmon Oncorhynchus gorbuscha)—was assessed by means of direct infusion to quadrupole time-of-flight mass spectrometry. Of the 2056 distinct m/z features detected by a mass spectrometry metabolic profiling of blood plasma samples, fifteen metabolites in the classes of dipeptides, fatty acids, glycerolipids, phosphoethanolamines and phosphatidylcholines were significantly associated with ageing rate, independent of species differences. This is the first study of the metabolome of fishes with various ageing rate, and this untargeted approach highlighted the metabolic conditions that may serve to assess the ageing process.