Exogenous and endogenous corticosterone in feathers

In birds, the steroid hormone corticosterone (CORT) increases in response to real or perceived threats to homeostasis. A long‐term record of CORT exposure is recorded in feathers when the hormone is incorporated into the keratinized tissue, and then preserved when the mature feather is cut off from the blood supply. The opportunity to retrospectively assess the exposure of an individual to stressors by measuring the amount of CORT in a feather has generated excitement amongst avian ecologists. However, this technique is relatively new and requires additional validations. In this study, we performed experiments in wild caught European starlings Sturnus vulgaris to test whether: 1) CORT deposition in the feather depends on time of day and 2) whether an ecologically relevant stressor (unpredictable food access) causes a change in feather CORT. We found that exogenous CORT was incorporated into feathers during the day and the night. However, there was no difference in feather CORT between birds with unpredictable access to food and those with continuous access, indicating that feather CORT might not always detect ecologically relevant stressors.     

¹⁸F]GE-180: a novel fluorine-18 labelled PET tracer for imaging Translocator protein 18 kDa (TSPO)

A series of tricyclic compounds have been synthesised and evaluated in vitro for affinity against Translocator protein 18 kDa (TSPO) and for preferred imaging properties. The most promising of the compounds were radiolabelled and evaluated in vivo to determine biodistribution and specificity for high expressing TSPO regions. Metabolite profiling in brain and plasma was also investigated. Evaluation in an autoradiography model of neuroinflammation was also carried out for the best compound, 12a ([(18)F]GE-180).     

Ca2+-Atpase inhibitor,cyclopiazonic acid,releases Ca2+ from intracellular stores in rbl-2h3 mast cells and activates a Ca2+ influx pathway that is permeable to sodium and manganese

Cyclopiazonic acid has been reported to inhibit the Ca²⁺-ATPase of intracellular calcium stores in some nonexcitable cell types, such as myeloid cells and lymphocytes. The present study examines the effects of cyclopizonic acid on rat basophilic leukemia (RBL) cells, a mucosal mast cell line. Addition of cyclopiazonic acid to fura-2-loaded RBL cells evoked a biphasic increase in free ionized intracellular calcium. Release of stored calcium accounted for the first phase of this response. The second phase was determined to be calcium entering through an influx pathway activated by cyclopiazonic acid. The influx pathway was selective for calcium, But was somewhat permeable to manganese. However, in a Ca²⁺-free solution containing EGTA, sodium ions permeated freely. This influx pathway appears to be identical to that which is activated by antigen, the physiological stimulus to the cells. Cyclopiazonic acid also induced secretion when combined with the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate, which activates protein kinae C. © 1995 Wiley-Liss, Inc.     

The inhibitory region of troponin-I alters the ability of F-actin to interact with different segments of myosin.

Peptides corresponding to the N-terminus of skeletal myosin light chain 1 (rsMLC1 1-37) and the short loop of human cardiac beta-myosin (hcM398-414) have been shown to interact with skeletal F-actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128-153) to F-actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161-181, all dissociate from F-actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F-actin. It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that interaction at different locations of F-actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

Polyethylene glycol as a hydration agent in oriented membrane bilayer samples

Techniques such as NMR, ESR, fluorescence depolarization, and neutron scattering are commonly used to investigate the physical properties of membranes. Oriented membrane bilayer systems (single crystals) are often employed in these investigations. It is important to know and be able to control the level of hydration in these samples. In particular, one must have confidence that a sample is in fact “fully hydrated” and remains so during the course of the experiment. Full hydration is difficult to obtain by hydrating oriented samples using water-saturated vapor. An alternative method for hydrating oriented samples is to surround the oriented sample by a polymer solution. Higher hydration levels are achieved using this method. Three nuclear magnetic resonance studies using headgroup deuterated 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) were done to compare the hydration level of oriented headgroup samples surrounded by a polymer/water solution and fully hydrated multibilayer dispersions. Transition temperatures, quadrupolar splittings (at 50°C) and spin-lattice relaxation times (at 50°C) were measured. The simple tests of the transition temperature and quadrupolar splitting to determine full hydration, as my results show, are not sufficient. In this paper I demonstrate that more fully hydrated samples can easily be achieved by surrounding the oriented sample with a 5 wt% polyethylene glycol/water solution than by hydrating in water saturated vapor.     

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.     

Gas chromatographic—mass spectrometric characterisation of some novel hydroxyeicosatetraenoic acids formed on incubation of arachidonic acid with microsomes from induced rat livers

The biotransformation of arachidonic acid by rat liver microsomes from both control animals and animals pretreated with known inducers of cytochrome P-450 isoenzymes has been studied using a combination of reversed- and normal-phase high-performance liquid chromatography and combined gas chromatography—electron-impact mass spectrometry. The metabolite profiles observed were found to be dependent upon the inducing agent. Five metabolites were identified, namely 16-, 17-, 18-, 19- and 20-hydroxylated arachidonic acids. Of these the 16- and 17-isomers have not been reported as products of arachidonic acid metabolism by any biological system and the 18-isomer has not been reported as a product of liver metabolism.